Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
  • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

• the present disclosure relates generally to the use of nucleic acids to treat hearing loss in a human subject.
• Hearing loss can be conductive (arising from the ear canal or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed.
• Most forms of syndromic and/or non-syndromic deafness are associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural deafness), although some forms may involve changes in the middle ear (conductive hearing loss).
• the great majority of human sensorineural hearing loss is caused by abnormalities in the hair cells of the organ of Corti in the cochlea (poor hair cell function). The hair cells may be abnormal at birth, or may be damaged during the lifetime of an individual (e.g., as a result of noise trauma or infection).
• the present disclosure provides the recognition that some diseases or conditions associated with hearing loss can be treated via, e.g., replacement and/or addition of certain gene products.
• the present disclosure further provides that gene products involved in the
• a gene product can be encoded by a gene encoding a secreted target protein (e.g., an NDP gene, or a heat shock protein (HSP) gene, e.g., an HSPA1 A gene) or a characteristic portion thereof.
• a gene product can be a secreted target protein or a characteristic portion thereof.
• AAV particles can be useful for administration of compositions that result in expression of gene products involved in the development, function, and/or maintenance of inner ear cells, and/or the treatment of hearing loss, or diseases or conditions associated with hearing loss.
• AAV particles comprise (i) a AAV polynucleotide construct (e.g., a recombinant AAV polynucleotide construct), and (ii) a capsid comprising capsid proteins.
• an AAV polynucleotide construct comprises a gene encoding a secreted target protein (e.g., an NDP gene or a heat shock protein (HSP) gene, e.g., an HSPA1 A gene or a DNJB gene) or a characteristic portion thereof.
• a secreted target protein e.g., an NDP gene or a heat shock protein (HSP) gene, e.g., an HSPA1 A gene or a DNJB gene
• HSP heat shock protein
• AAV particles of the present disclosure are referred to as recombinant AAV particles or rAAV particles.
• compositions including a single AAV vector, wherein the single AAV vector comprises a nucleic acid sequence that encodes a secreted target protein.
• the single AAV vector comprises a nucleic acid sequence that encodes a secreted target protein.
• a nucleic acid encoding a secretion signal sequence operatively linked to the secreted target protein is generated at the locus of the secreted target protein.
• the primate cell expresses and secretes the secreted target protein.
• compositions comprising polynucleotide constructs comprising a gene encoding a secreted target protein (e.g., an NDP gene or a heat shock protein (HSP) gene, e.g., an HSPA1 A gene and a DNJB gene) or a characteristic portion thereof.
• a construct may further include regulatory elements operably attached to a coding sequence.
• included regulatory elements facilitate tissue specific expression at physiologically suitable levels.
• administration involves surgical intervention and the delivery of AAV particles comprising therapeutic constructs.
• AAV particles may be delivered to the inner ear of a subject in need thereof by surgical introduction through the round window membrane.
• efficacy of an intervention is determined through established tests, and measurements are compared to known control measurements.
• a single AAV vector further includes a 5’ untranslated region (UTR), a 3’ UTR, or both.
• UTR untranslated region
• a secreted target protein is norrin cysteine knot growth factor (NDP).
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a nucleic acid that encodes a secreted target protein includes a sequence that is at least 80% identical (e.g., at least 82%o, at least 84%, at least 86%, at least 88%>, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%>, at least 97%, at least 98%, at least 99%>, or 100%) to SEQ ID NO: 2.
• a nucleic acid that encodes a secreted target protein includes a sequence that is at least 90% identical (e.g., at least 92%, at least 94%>, at least 95%, at least 96%o, at least 97%, at least 98%, at least 99%>, or 100%) to SEQ ID NO: 2.
• a nucleic acid that encodes a secreted target protein includes a sequence that is at least 99% identical to SEQ ID NO: 2.
• a secreted target protein is a heat shock protein.
• a heat shock protein is a heat shock protein family A (Hsp70) member 1A (HSPA1A).
• a heat shock protein is a heat shock protein 40 (Hsp40)/DNJ family member, e.g., Hsp40.
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a nucleic acid that encodes a secreted target protein includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 4.
• a nucleic acid that encodes a secreted target protein includes a sequence that is at least 90% identical (e.g., at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 4.
• a nucleic acid that encodes a secreted target protein includes a sequence that is at least 99% identical to SEQ ID NO: 4.
• an AAV vector further includes one or both of a promoter and a Kozak sequence.
• an AAV vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
• an AAV vector further includes a poly(dA) sequence.
• a secretion signal sequence includes SEQ ID NO: 5.
• a sequence encoding a secretion signal sequence includes
• a secretion signal sequence includes SEQ ID NO: 7.
• a sequence encoding the secretion signal sequence includes SEQ ID NO: 8.
• a single AAV vector includes a sequence that is at least
• a single AAV vector includes a sequence that is at least
• a single AAV vector includes a sequence that is at least
• a single AAV vector includes a sequence that is at least
• a single AAV vector includes a sequence that is at least
• a single AAV vector includes a sequence that is at least
• compositions described herein further includes a pharmaceutically acceptable excipient.
• kits including a composition (e.g., any of the compositions described herein).
• a composition is pre- loaded in a syringe.
• compositions include introducing into an inner ear of a mammal a therapeutically effective amount of a composition (e.g., any of the compositions described herein).
• a mammal is a human.
• a mammal has been previously identified as having a defective secreted target gene.
• methods of increasing expression of a full-length secreted target protein in a mammalian cell that include introducing a composition (e.g., any of the compositions described herein) into the mammalian cell.
• a mammalian cell is a cochlear inner hair cell, a supporting cell, a ganglion cell, a clear cell, a cuboidal cell, a cartilage cell, a cell of the tegmentum vasculosum, a homogene cell, a Hensen’s cell, a Deiters’ cell, a pillar cell, or a border cell.
• a mammalian cell is a human cell.
• a mammalian cell has previously been determined to have a defective secreted target gene.
• Also provided herein are methods of increasing the level of a full-length secreted target protein in an inner ear of a mammal that include: introducing into the inner ear of a mammal a therapeutically effective amount of a composition (e.g., any of the compositions described herein).
• a mammal has been previously identified as having a defective secreted target gene.
• a mammal is a human.
• a composition e.g., any of the compositions described herein
• a subject is a human.
• a method further includes, prior to the administering step, determining that the subject has a defective secreted target gene.
• methods of treating or preventing hearing loss in a subject identified as having a defective NDP gene that include: administering a therapeutically effective amount of a composition (e.g., any of the compositions described herein) into an inner ear of the subject.
• a subject has been identified or diagnosed as having Nome disease pseudoglioma.
• a subject is a human.
• a method further includes, (e.g., prior to the administering step) determining that the subject has a defective NDP gene.
• compositions including at least two different nucleic acid vectors, wherein: each of the at least two different vectors includes a coding sequence that encodes a different portion of a secreted target protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes a full-length secreted target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the secreted target protein genomic DNA, and lacks an intronic sequence between the two neighboring exons; and when introduced into a mammalian cell the at least two different vectors undergo concatamerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a secretion signal sequence operatively linked to a full-
• each of at least two different vectors is a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral vector.
• each of at least two different vectors is a human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or a Pl-derived artificial chromosome (PAC).
• each of at least two different vectors is a viral vector selected from an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector.
• AAV adeno-associated virus
• each of at least two different vectors is an AAV vector.
• the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions.
• the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different encoded portion.
• the overlapping amino acid sequence is between about 30 amino acid residues to about 600 amino acid residues in length.
• vectors include two different vectors, each of which includes a different segment of an intron, wherein the intron includes the nucleotide sequence of an intron that is present in the secreted target protein genomic DNA, and wherein the two different segments overlap in sequence by at least 100 nucleotides.
• two different segments overlap in sequence by about 100 nucleotides to about 800 nucleotides (e.g., any of the subranges therein).
• the nucleotide sequence of each of the at least two different vectors is between about 500 nucleotides to about 10,000 nucleotides in length (e.g., any of the subranges therein).
• the nucleotide sequence of each of the at least two different vectors is between about 500 nucleotides to about 5,000 nucleotides in length (e.g., any of the subranges therein).
• the number of different vectors in the composition is two.
• a first of the two different vectors includes a coding sequence that encodes an N-terminal portion of the secreted target protein.
• an N-terminal portion of the secreted target protein is between about 30 amino acids to about 600 amino acids in length (e.g., any of the subranges therein).
• an N-terminal portion of the secreted target protein is between about 100 amino acids to about 500 amino acids in length (e.g., any of the subranges therein).
• a first vector further includes one or both of a promoter and a Kozak sequence.
• a first vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
• a second of the two different vectors includes a coding sequence that encodes a C-terminal portion of the secreted target protein.
• a C-terminal portion of the secreted target protein is between about 30 amino acids to about 600 amino acids in length (e.g., any of the subranges therein).
• a C-terminal portion of the secreted target protein is between about 200 amino acids to about 500 amino acids in length (e.g., any of the subranges therein).
• a second vector further includes a poly(dA) sequence.
• a first vector, a second vector, or both vectors further includes a 5’ untranslated region (UTR), a 3’ UTR, or both.
• a secreted target protein is norrin cysteine knot growth factor (NDP).
• NDP norrin cysteine knot growth factor
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein is a heat shock protein.
• a heat shock protein is a heat shock protein family A (Hsp70) member 1A (HSPA1A).
• a heat shock protein is a heat shock protein 40 (Hsp40)/DNJ family member, e.g., Hsp40.
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secretion signal sequence includes SEQ ID NO: 5.
• a secretion signal sequence includes SEQ ID NO: 7.
• compositions including two different nucleic acid vectors, wherein: a first nucleic acid vector of the two different nucleic acid vectors includes a promoter, a first coding sequence that encodes an N-terminal portion of an secreted target protein positioned 3’ of the promoter, and a splicing donor signal sequence positioned at the 3’ end of the first coding sequence; and a second nucleic acid vector of the two different nucleic acid vectors includes a splicing acceptor signal sequence, a second coding sequence that encodes a C- terminal portion of an secreted target protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the amino acid sequences of the encoded portions do not overlap, wherein no single vector of the two different vectors encodes a full-length secreted target protein, and
• a coding sequence of at least one of the vectors includes a nucleotide sequence spanning two neighboring exons of secreted target genomic DNA, and lacks an intronic sequence between the two neighboring exons.
• compositions including: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of an secreted target protein positioned 3’ of the promoter, a splicing donor signal sequence positioned at the 3’ end of the first coding sequence, and a first detectable marker gene positioned 3’ of the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second detectable marker gene, a splicing acceptor signal sequence positioned 3’ of the second detectable marker gene, a second coding sequence that encodes a C-terminal portion of an secreted target protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the respective amino acid sequences of the encoded portions do not overlap with each
• a coding sequence of at least one of the vectors includes a nucleotide sequence spanning two neighboring exons of secreted target genomic DNA, and lacks an intronic sequence between the two neighboring exons.
• a first or second detectable marker gene encodes alkaline phosphatase.
• a first and second detectable marker genes are the same.
• compositions including: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of an secreted target protein positioned 3’ to the promoter, a splicing donor signal sequence positioned at the 3’ end of the first coding sequence, and a FI phage recombinogenic region positioned 3’ to the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second FI phage recombinogenic region, a splicing acceptor signal sequence positioned 3’ of the second FI phage recombinogenic region, a second coding sequence that encodes a C-terminal portion of an secreted target protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in
• a coding sequence of at least one of the vectors includes a nucleotide sequence spanning two neighboring exons of secreted target genomic DNA, and lacks an intronic sequence between the two neighboring exons.
• a first vector, the second vector, or both vectors further includes a 5’ untranslated region (UTR), a 3’ UTR, or both.
• a secreted target protein is norrin cysteine knot growth factor (NDP).
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein is a heat shock protein.
• a heat shock protein is a heat shock protein family A (Hsp70) member 1A (HSPA1A).
• a heat shock protein is a heat shock protein 40 (Hsp40)/DNJ family member, e.g., Hsp40.
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secreted target protein includes a sequence that is at least
• a secretion signal sequence includes SEQ ID NO: 5.
• a secretion signal sequence includes SEQ ID NO: 7.
• compositions described herein further includes a pharmaceutically acceptable excipient.
• kits including a composition (e.g., any of the compositions described herein).
• kits further includes a pre-loaded syringe including the composition.
• kits that include: introducing into an inner ear of a mammal a therapeutically effective amount of a composition (e.g., any of the compositions described herein).
• a mammal is a human.
• a mammal has been previously identified as having a defective secreted target gene.
• kits for increasing expression of a full-length secreted target protein in a mammalian cell that include: introducing a composition (e.g., any of the compositions described herein) into the mammalian cell.
• a composition e.g., any of the compositions described herein
• a mammalian cell is a cochlear inner hair cell, a supporting cell, a ganglion cell, a clear cell, a cuboidal cell, a cartilage cell, a cell of the tegmentum vasculosum, a homogene cell, a Hensen’s cell, a Deiters’ cell, a pillar cell, or a border cell.
• a mammalian cell is a human cell.
• a mammalian cell has previously been determined to have a defective secreted target gene.
• methods of increasing the level of a full-length secreted target protein in an inner ear of a mammal that include: introducing into the inner ear of the mammal a therapeutically effective amount of a composition (e.g., any of the compositions described herein).
• a mammal has been previously identified as having a defective secreted target gene.
• a mammal is a human.
• a composition e.g., any of the compositions described herein
• a subject is a human.
• a method further includes prior to the administering step, determining that the subject has a defective secreted target gene.
• a composition e.g., any of the compositions described herein
• a subject has been identified or diagnosed as having Nome disease pseudoglioma.
• a subject is a human.
• a method further includes prior to the administering step, determining that the subject has a defective NDP gene.
• methods of treating or preventing vision loss in a subject identified as having a defective NDP gene that include: administering a therapeutically effective amount of a composition (e.g., any of the compositions described herein) into an inner ear or central nervous system of the subject, or systemically administering a therapeutically effective amount of a composition (e.g., any of the compositions described herein) to the subject.
• a subject is a human.
• a method further includes prior to the administering step, determining that the subject has a defective NDP gene.
• Also provided herein are methods of treating or preventing vision loss in a subject identified as having a defective NDP gene that include: administering a therapeutically effective amount of a composition (e.g., any of the compositions described herein) into an inner ear or central nervous system of the subject, or systemically administering a therapeutically effective amount of a composition (e.g., any of the compositions described herein) to the subject.
• a composition e.g., any of the compositions described herein
• a subject is a human.
• a method further includes prior to the administering step, determining that the subject has a defective NDP gene.
• the term“a” and“an” refers to one or to more than one (i.e., at least one) of the grammatical object of the article.
• “an element” encompasses one element and more than one element. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process.
• a polynucleotide or polypeptide is represented by a sequence of letters (e.g., A, C, G, and T, which denote adenosine, cytidine, guanosine, and thymidine, respectively in the case of a polynucleotide), such polynucleotides or polypeptides are presented in 5’ to 3’ or N-terminus to C-terminus order, from left to right.
• Administration typically refers to administration of a composition to a subject or system to achieve delivery of an agent to a subject or system.
• an agent is, or is included in, a composition; in some embodiments, an agent is generated through metabolism of a composition or one or more components thereof.
• routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
• administration may be systematic or local.
• a systematic administration can be intravenous.
• administration can be local. Local administration can involve delivery to cochlear perilymph via, e.g., injection through a round-window membrane or into scala-tympani, a scala-media injection through endolymph, perilymph and/or endolymph following canalostomy.
• administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
• Allele refers to one of two or more existing genetic variants of a specific polymorphic genomic locus.
• Amelioration refers to prevention, reduction or palliation of a state, or improvement of a state of a subject. Amelioration may include, but does not require, complete recovery or complete prevention of a disease, disorder or condition.
• amino acid refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
• an amino acid has a general structure, e.g., H2N-C(H)(R)-COOH.
• an amino acid is a naturally- occurring amino acid.
• an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid.
• Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
• Nonstandard amino acid refers to any amino acid, other than standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
• an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with general structure as shown above.
• an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and/or a hydroxyl group) as compared with a general structure.
• such modification may, for example, alter circulating half-life of a polypeptide containing a modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
• such modification does not significantly alter a relevant activity of a polypeptide containing a modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
• the terms“approximately” or“about” may be applied to one or more values of interest, including a value that is similar to a stated reference value.
• the term“approximately” or“about” refers to a range of values that fall within ⁇ 10% (greater than or less than) of a stated reference value unless otherwise stated or otherwise evident from context (except where such number would exceed 100% of a possible value).
• the term“approximately” or “about” may encompass a range of values that within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a reference value.
• the term“associated” describes two events or entities as“associated” with one another, if the presence, level and/or form of one is correlated with that of the other.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
• two or more entities are physically“associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
• two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof
• biologically active refers to an observable biological effect or result achieved by an agent or entity of interest.
• a specific binding interaction is a biological activity.
• modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity.
• presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.
• Characteristic portion refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance.
• a characteristic portion of a substance is a portion that is found in a given substance and in related substances that share a particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity.
• a characteristic portion shares at least one functional characteristic with the intact substance.
• a“characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide.
• each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids.
• a characteristic portion of a substance e.g., of a protein, antibody, etc.
• a characteristic portion may be biologically active.
• Characteristic sequence As used herein, the term“characteristic sequence” is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.
• Characteristic sequence element As used herein, the phrase“characteristic sequence element” refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, presence of a characteristic sequence element correlates with presence or level of a particular activity or property of a polymer. In some embodiments, presence (or absence) of a
• characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers.
• a characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,
• a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share a sequence element.
• Combination therap refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously. In some embodiments, two or more agents may be
• two or more agents may be administered in overlapping dosing regimens.
• Comparable refers to two or more agents, entities, situations, sets of conditions, subjects, populations, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
• comparable sets of agents, entities, situations, sets of conditions, subjects, populations, etc. are characterized by a plurality of substantially identical features and one or a small number of varied features.
• Construct refers to a composition including a polynucleotide capable of carrying at least one heterologous polynucleotide.
• a construct can be a plasmid, a transposon, a cosmid, an artificial chromosome (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a Pl-derived artificial chromosome (PAC)) or a viral construct, and any Gateway® plasmids.
• HAC human artificial chromosome
• YAC yeast artificial chromosome
• BAC bacterial artificial chromosome
• PAC Pl-derived artificial chromosome
• a construct can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host primate cell or in an in vitro expression system.
• a construct may include any genetic element (e.g., a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral construct, etc.) that is capable of replicating when associated with proper control elements.
• “construct” may include a cloning and/or expression construct and/or a viral construct (e.g., an adeno-associated virus (AAV) construct, an adenovirus construct, a lentivirus construct, or a retrovirus construct).
• AAV adeno-associated virus
• the term“vector” refers to a construct.
• conservative amino acid substitution refers to instances describing a conservative amino acid substitution, including a substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity).
• a conservative amino acid substitution will not substantially change functional properties of interest of a protein, for example, ability of a receptor to bind to a ligand.
• Examples of groups of amino acids that have side chains with similar chemical properties include: aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (lie, I); aliphatic-hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gin, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine (Cys, C) and methi
• Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine (Val/Leu/Ile, V/L/I), phenylalanine/tyrosine (Phe/Tyr, F/Y), lysine/arginine (Lys/Arg, K/R), alanine/valine (Ala/Val, A/V), glutamate/aspartate (Glu/Asp, E/D), and asparagine/glutamine (Asn/Gln, N/Q).
• a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis.
• a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, G.H. et al., 1992, Science 256: 1443-1445, which is incorporated herein by reference in its entirety.
• a substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.
• control refers to the art-understood meaning of a“control” being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables.
• a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. For example, in one experiment, a“test” (i.e., a variable being tested) is applied. In a second experiment, a“control,” the variable being tested is not applied.
• a control is a historical control (e.g., of a test or assay performed previously, or an amount or result that is previously known).
• a control is or comprises a printed or otherwise saved record. In some embodiments, a control is a positive control. In some embodiments, a control is a negative control.
• Endogenous In general, as used herein, the term“endogenous” refers to any material originating from within an organism, cell, or tissue.
• Engineered refers to an aspect of having been manipulated by the hand of man.
• a cell or organism is considered to be“engineered” if it has been manipulated so that its genetic information is altered (e.g, new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
• new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols.
• progeny of an engineered polynucleotide or cell are typically still referred to as“engineered” even though the actual manipulation was performed on a prior entity.
• Excipient refers to an inactive (e.g., non- therapeutic) agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
• suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• expression refers to generation of any gene product (e.g., transcript, e.g., mRNA, e.g., polypeptide, etc.) from a nucleic acid sequence.
• a gene product can be a transcript.
• a gene product can be a polypeptide.
• expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
• the term“expression” refers to the transcription and/or translation of a particular nucleotide sequence encoding a protein.
• exogenous refers to any material introduced from or originating from outside an organism, cell, or tissue that is not produced or does not originate from the same organism, cell, or tissue in which it is being introduced.
• a“functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
• a functional biological molecule is characterized relative to another biological molecule which is non-functional in that the“non-functional” version does not exhibit the same or equivalent property and/or activity as the“functional” molecule.
• a biological molecule may have one function, two functions (i.e., bifunctional) or many functions (i.e., multifunctional).
• Gene refers to a DNA sequence in a
• chromosome that codes for a gene product (e.g., an RNA product, e.g., a polypeptide product).
• a gene product e.g., an RNA product, e.g., a polypeptide product.
• a gene includes coding sequence (i.e., sequence that encodes a particular product). In some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc ).
• regulatory sequences e.g., promoters, enhancers, etc.
• the term“gene” generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude application of the term“gene” to non-protein-coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a polypeptide-coding nucleic acid.
• a gene may encode a polypeptide, but that polypeptide may not be functional, e g., a gene variant may encode a polypeptide that does not function in the same way, or at all, relative to the wild-type gene.
• a gene may encode a transcript which, in some embodiments, may be toxic beyond a threshold level. In some embodiments, a gene may encode a polypeptide, but that polypeptide may not be functional and/or may be toxic beyond a threshold level.
• hearing loss may be used to a partial or total inability of a living organism to hear.
• hearing loss may be acquired.
• hearing loss may be hereditary.
• hearing loss may be genetic.
• hearing loss may be as a result of disease or trauma (e.g., physical trauma, treatment with one or more agents resulting in hearing loss, etc.).
• hearing loss may be due to one or more known genetic causes and/or syndromes.
• hearing loss may be of unknown etiology.
• hearing loss may or may not be mitigated by use of hearing aids or other treatments.
• heterologous may be used in reference to one or more regions of a particular molecule as compared to another region and/or another molecule.
• heterologous polypeptide domains refers to the fact that polypeptide domains do not naturally occur together (e.g., in the same polypeptide).
• a polypeptide domain from one polypeptide may be fused to a polypeptide domain from a different polypeptide.
• two polypeptide domains would be considered“heterologous” with respect to each other, as they do not naturally occur together.
• Identity refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
• polymeric molecules are considered to be“substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
• Calculation of percent identity of two nucleic acid or polypeptide sequences can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
• aligning two sequences for optimal comparison purposes e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes.
• a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; nucleotides at corresponding positions are then compared.
• a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as a corresponding position in the second sequence, then the two molecules (i.e., first and second) are identical at that position.
• Percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
• an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
• an appropriate reference is a negative reference; in some embodiments, an appropriate reference is a positive reference.
• Isolated means altered or removed from the natural state.
• a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is“isolated”
• An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
• nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
• a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
• “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
• a“nucleic acid” is or comprises RNA; in some embodiments, a“nucleic acid” is or comprises DNA.
• a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
• a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
• a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
• a nucleic acid has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds.
• a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine).
• adenosine thymidine, guanosine, cytidine
• uridine deoxyadenosine
• deoxythymidine deoxy guanosine
• deoxycytidine deoxycytidine
• a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases
• a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
• a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
• a nucleic acid includes one or more introns.
• nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
• a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
• a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
• a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is complementary to a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
• the term“nucleic acid” or“polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides.
• nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.
• Operably linked refers to a juxtaposition wherein the
• control element“operably linked” to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element.
• “operably linked” control elements are contiguous (e.g., covalently linked) with coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.
• “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
• a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
• a functional linkage may include transcriptional control.
• a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
• composition refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.
• an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
• a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for, e.g., administration, for example, an injectable formulation that is, e.g., an aqueous or non- aqueous solution or suspension or a liquid drop designed to be administered into an ear canal.
• a pharmaceutical composition may be formulated for administration via injection either in a particular organ or compartment, e.g., directly into an ear, or systemic, e.g., intravenously.
• a formulation may be or comprise drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, capsules, powders, etc.
• an active agent may be or comprise an isolated, purified, or pure compound.
• composition as disclosed herein, means that a carrier, diluent, or excipient is compatible with other ingredients of a composition and not deleterious to a recipient thereof.
• composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting a subject compound from one organ, or portion of a body, to another organ, or portion of a body.
• Each carrier must be is“acceptable” in the sense of being compatible with other ingredients of a formulation and not injurious to a patient.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
• powdered tragacanth malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.
• oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
• polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
• mRNA messenger RNA
• a 3’ poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
• a poly(A) tail can be added onto transcripts that contain a specific sequence, the polyadenylation signal or“poly(A) sequence.”
• a poly(A) tail and proteins bound to it aid in protecting mRNA from degradation by exonucleases.
• Polyadenylation can be affect transcription termination, export of the mRNA from the nucleus, and translation. Typically, polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain can be cleaved through the action of an endonuclease complex associated with RNA polymerase.
• the cleavage site can be characterized by the presence of the base sequence AAUAAA near the cleavage site.
• adenosine residues can be added to the free 3’ end at the cleavage site.
• a“poly(A) sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the additional of a series of adenosines to the 3’ end of the cleaved mRNA.
• Polypeptide refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds.
• a polypeptide has an amino acid sequence that occurs in nature.
• a polypeptide has an amino acid sequence that does not occur in nature.
• a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
• a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
• a polypeptide may include one or more pendant groups or other modifications, e g., modifying or attached to one or more amino acid side chains, at a polypeptide’s N-terminus, at a polypeptide’s C-terminus, or any combination thereof.
• pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
• polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art.
• useful modifications may be or include, e.g., terminal acetylation, amidation, methylation, etc.
• a protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
• the term“peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
• a protein is antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
• polynucleotide refers to any polymeric chain of nucleic acids.
• a polynucleotide is or comprises RNA; in some embodiments, a polynucleotide is or comprises DNA.
• a polynucleotide is, comprises, or consists of one or more natural nucleic acid residues.
• a polynucleotide is, comprises, or consists of one or more nucleic acid analogs.
• a polynucleotide analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a
• a polynucleotide has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds.
• a polynucleotide is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine).
• a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, inter
• a polynucleotide comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
• a polynucleotide has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
• a polynucleotide includes one or more introns.
• a polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by
• a polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
• a polynucleotide is partly or wholly single stranded; in some embodiments, a polynucleotide is partly or wholly double stranded. In some embodiments, a polynucleotide has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.
• a polynucleotide has enzymatic activity.
• a“nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.
• Modifications can be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, and histidine
• acidic side chains e.g., aspartic acid and glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine
• beta-branched side chains e.g., threonine, valine, and isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, and histidine
• Protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
• Recombinant is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression construct transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that
• one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).
• Reference describes a standard or control relative to which a comparison is performed.
• an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
• a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
• a reference or control is a historical reference or control, optionally embodied in a tangible medium.
• a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
• a reference is a negative control reference; in some embodiments, a reference is a positive control reference.
• regulatory element refers to non-coding regions of DNA that regulate, in some way, expression of one or more particular genes. In some embodiments, such genes are apposed or “in the neighborhood” of a given regulatory element. In some embodiments, such genes are located quite far from a given regulatory element. In some embodiments, a regulatory element impairs or enhances transcription of one or more genes. In some embodiments, a regulatory element may be located in cis to a gene being regulated. In some embodiments, a regulatory element may be located in trans to a gene being regulated.
• a regulatory sequence refers to a nucleic acid sequence which is regulates expression of a gene product operably linked to a regulatory sequence.
• this sequence may be an enhancer sequence and other regulatory elements which regulate expression of a gene product.
• sample typically refers to an aliquot of material obtained or derived from a source of interest.
• a source of interest is a biological or environmental source.
• a source of interest may be or comprise a cell or an organism, such as a microbe (e.g., virus), a plant, or an animal ( e.g ., a human).
• a source of interest is or comprises biological tissue or fluid.
• a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component s) thereof.
• a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
• a biological fluid may be or comprise a plant exudate.
• a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchi oalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
• a biological sample is or comprises cells obtained from an individual.
• a sample is a“primary sample” obtained directly from a source of interest by any appropriate means.
• sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
• a“processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
• Subject refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms).
• a subject is suffering from a relevant disease, disorder or condition.
• a subject is susceptible to a disease, disorder, or condition.
• a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
• a subject does not display any symptom or characteristic of a disease, disorder, or condition.
• a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
• a subject is a patient.
• a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
• the subject is a rodent (e.g., a rat or mouse), a rabbit, a sheep, a goat, a pig, a dog, a cat, a non-human primate, or a human.
• the subject has or is at risk of hearing loss and/or vision loss.
• the subject has been previously identified as having a mutation in a secreted target gene (e.g., a NDP gene or a HSPA1A gene). In some embodiments, the subject has been identified as having a mutation in a secreted target gene (e.g., a NDP gene or a
• HSPA1 A gene has been diagnosed with hearing loss and/or vision loss.
• the subject has been identified as having hearing loss and/or vision loss.
• Substantially refers to a qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
• biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
• the term“substantially” is therefore used herein to capture a potential lack of completeness inherent in many biological and chemical phenomena.
• transfected refers to a process by which exogenous nucleic acid is transferred or introduced into a cell.
• A“transfected,”“transformed,” or “transduced” mammalian cell is one that has been transfected, transformed or transduced with exogenous nucleic acid.
• Transient expression refers to the expression of a non-integrated coding sequence for a short period of time (e.g., hours or days).
• the coding sequence that is transiently expressed in a cell e.g., a mammalian cell
• treatment refers to any administration of a therapy that partially or completely alleviates, ameliorates, eliminates, reverses, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
• such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
• such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
• treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of a given disease, disorder, and/or condition.
• a treatment is“therapeutically effective” when it results in a reduction in one or more of the number, severity, and frequency of one or more symptoms of a disease state (e.g., hearing loss or vision loss) in a subject (e.g., a human).
• a disease state e.g., hearing loss or vision loss
• a subject e.g., a human
• a therapeutically effective amount of a composition can result in an increase in the expression level of an active secreted target protein (e.g., an active NDP protein (e.g., a wildtype, full-length NDP protein or a variant of a NDP protein that has the desired activity) or an active HSPA1 A protein (e.g., a wildtype, full-length HSPA1 A protein or a variant of a HSPA1A protein that has the desired activity)) (e.g., as compared to the expression level prior to treatment with the composition).
• an active secreted target protein e.g., an active NDP protein (e.g., a wildtype, full-length NDP protein or a variant of a NDP protein that has the desired activity)
• an active HSPA1 A protein e.g., a wildtype, full-length HSPA1 A protein or a variant of a HSPA1A protein that has the desired activity
• a therapeutically effective amount of a composition can result in an increase in the expression level of an active secreted target protein (e.g., an active NDP protein (e.g., a wildtype, full-length NDP protein or an active variant) or an active HSPA1 A protein (e.g., a wildtype, full-length HSPA1 A protein or a variant of a HSPA1 A protein that has the desired activity) or an active heat shock protein (e.g., a wildtype, full-length heat shock protein or a variant of a heat shock protein that has the desired activity)) in a target cell (e.g., a cochlear inner hair cell).
• an active secreted target protein e.g., an active NDP protein (e.g., a wildtype, full-length NDP protein or an active variant) or an active HSPA1 A protein (e.g., a wildtype, full-length HSPA1 A protein or a variant of a HSPA1 A protein that has the desired activity)
• an active secreted target protein e.g., an active NDP protein (e.g., a wildtype, full-length NDP protein or active variant) or an active HSPA1A protein (e.g., a wildtype, full-length HSPA1 A protein or a variant of a HSPA1 A protein that has the desired activity) or an active heat shock protein (e.g., a wildtype, full-length heat shock protein or a variant of a heat shock protein that has the desired activity)), and/or an increase in one or more activities of a secreted target protein (e.g., a NDP protein or a HSPA1 A protein or a Hsp40 protein) in a target cell (e.g., as compared to a reference level, such as the level (s) in a subject prior to treatment, the level(s) in a subject having a mutation in a NDP gene or a HSPA1 A gene, or the level(
• Variant refers to a version of something, e.g., a gene sequence, that is different, in some way, from another version.
• a reference version is typically chosen and a variant is different relative to that reference version.
• a variant can have the same or a different (e.g., increased or decreased) level of activity or functionality than a wild type sequence.
• a variant can have improved functionality as compared to a wild-type sequence if it is, e.g., codon-optimized to resist degradation, e.g., by an inhibitory nucleic acid, e.g., miRNA.
• a variant has a reduction or elimination in activity or functionality or a change in activity that results in a negative outcome (e.g., increased electrical activity resulting in chronic depolarization that leads to cell death).
• a variant is referred to herein as a loss-of-function variant.
• a NDP gene sequence is a wild-type sequence, which encodes a functional protein and exists in a majority of members of species with genomes containing the NDP gene.
• a gain-of-function variant can be a gene sequence of NDP that contains one or more nucleotide differences relative to a wild-type NDP gene sequence.
• a gain-of-function variant is a codon-optimized sequence which encodes a transcript or polypeptide that may have improved properties (e.g., less susceptibility to degradation, e.g., less susceptibility to miRNA mediated degradation) than its corresponding wild type (e.g., non-codon optimized) version.
• a loss-of- function variant has one or more changes that result in a transcript or polypeptide that is defective in some way (e.g., decreased function, non-functioning) relative to the wild type transcript and/or polypeptide.
• a mutation in a NDP sequence results in a non-functional or otherwise defective NDP protein.
• a HSPA1 A gene sequence is a wild-type sequence, which encodes a functional protein and exists in a majority of members of species with genomes containing the HSPA1A gene.
• a gain-of-function variant can be a gene sequence of HSPA1A that contains one or more nucleotide differences relative to a wild-type HSPA1A gene sequence.
• a DNAJB1 gene sequence is a wild- type sequence, which encodes a functional protein and exists in a majority of members of species with genomes containing the DNAJB1 gene.
• a gain-of-function variant can be a gene sequence of DNAJB 1 that contains one or more nucleotide differences relative to a wild-type DNAJB1 gene sequence.
• a DNAJB5 gene sequence is a wild-type sequence, which encodes a functional protein and exists in a majority of members of species with genomes containing the DNAJB5 gene.
• a gain-of-function variant can be a gene sequence of DNAJB5 that contains one or more nucleotide differences relative to a wild-type DNAJB5 gene sequence.
• a gain-of-function variant is a codon-optimized sequence which encodes a transcript or polypeptide that may have improved properties (e.g., less susceptibility to degradation, e.g., less susceptibility to miRNA mediated degradation) than its corresponding wild type (e.g., non-codon optimized) version.
• a loss-of-function variant has one or more changes that result in a transcript or polypeptide that is defective in some way (e.g., decreased function, non-functioning) relative to the wild type transcript and/or polypeptide.
• a mutation in a HSPA1 A sequence results in a non functional or otherwise defective HSPA1 A protein.
• Figure 1 is an exemplary nucleic acid vector, Construct 1 (SEQ ID NO: 9; 4185 bp) that includes an inverted terminal repeat (ITR) sequence (SEQ ID NO: 16), a CMV sequence (SEQ ID NO: 17), a CBA sequence (SEQ ID NO: 18), a chimeric intron sequence (SEQ ID NO: 19), a Norrin coding sequence (SEQ ID NO: 20), a 3’ untranslated region (UTR)-1023 sequence (SEQ ID NO: 22), a bovine growth hormone (bGH) poly(A) sequence (SEQ ID NO: 23), a hFVIII stuffer sequence (SEQ ID NO: 24), and an ITR sequence (SEQ ID NO: 30).
• ITR inverted terminal repeat
• SEQ ID NO: 16 CMV sequence
• CBA sequence SEQ ID NO: 18
• SEQ ID NO: 19 a chimeric intron sequence
• SEQ ID NO: 19 a Norrin coding sequence
• SEQ ID NO: 20
• Figure 2 is an exemplary nucleic acid vector, Construct 2 (SEQ ID NO: 10; 3662 bp) that includes an ITR sequence (SEQ ID NO: 16), a CMV sequence (SEQ ID NO: 17), a CBA sequence (SEQ ID NO: 18), a chimeric intron sequence (SEQ ID NO: 19), a Norrin coding sequence (SEQ ID NO: 20), a bGH poly(A) sequence (SEQ ID NO: 23), a hFVIII stuffer sequence (SEQ ID NO: 25), and an ITR sequence (SEQ ID NO: 30).
• ITR sequence SEQ ID NO: 16
• CMV sequence SEQ ID NO: 17
• CBA sequence SEQ ID NO: 18
• SEQ ID NO: 19 a chimeric intron sequence
• SEQ ID NO: 19 a Norrin coding sequence
• SEQ ID NO: 20 a bGH poly(A) sequence
• SEQ ID NO: 25 a hFVIII stuffer sequence
• Figure 3 is an exemplary nucleic acid vector, Construct 3 (SEQ ID NO: 11, 4444 bp), that includes an ITR sequence (SEQ ID NO: 16), a CMV sequence (SEQ ID NO: 17), a CBA sequence (SEQ ID NO: 18), a chimeric intron sequence (SEQ ID NO: 19), a Norrin coding sequence (SEQ ID NO: 21), a T2A sequence (SEQ ID NO: 26), a tGFP sequence (SEQ ID NO:
• Figure 4 is an exemplary nucleic acid vector, Construct 4 (SEQ ID NO: 12, 4468 bp), that includes an ITR sequence (SEQ ID NO: 16), a CMV sequence (SEQ ID NO: 17), a CBA sequence (SEQ ID NO: 18), a chimeric intron sequence (SEQ ID NO: 19), a Norrin coding sequence (SEQ ID NO: 21), a T2A sequence (SEQ ID NO: 26), an eGFP sequence (SEQ ID NO:
• Figure 5 is an exemplary nucleic acid vector, Construct 5 (SEQ ID NO: 13, 4764 bp), that includes an ITR sequence (SEQ ID NO: 16), a CMV sequence (SEQ ID NO: 17), a CBA sequence (SEQ ID NO: 18), a chimeric intron sequence (SEQ ID NO: 19), a 5’UTR-579 sequence (SEQ ID NO: 31), a Norrin coding sequence (SEQ ID NO: 20), a 3’ UTR-1023 sequence (SEQ ID NO: 22), a bGH poly(A) sequence (SEQ ID NO:23), a hFVIII stuffer sequence (SEQ ID NO: 24), and an ITR sequence (SEQ ID NO: 30).
• ITR sequence SEQ ID NO: 16
• CMV sequence SEQ ID NO: 17
• CBA sequence SEQ ID NO: 18
• a chimeric intron sequence SEQ ID NO: 19
• a 5’UTR-579 sequence SEQ ID NO: 31
• Figure 6 is an exemplary image of a Western blot showing harvested supernatant of HEK293FT cells transfected with plasmid 1, plasmid 2 or plasmid 3, and blotted with NDP antibody.
• Lane 1- prestained Page Ruler; Lane 2 - Construct 1; Lane 3 - Construct 2; Lane 4 - Construct 3; Lane 5 - untransfected control supernatant.
• Figure 7 is an exemplary fluorescent image showing Myo7a staining following ex vivo cochlea transduction with Anc80.NDP.UTR (Construct 1) or Anc80.NDP (Construct 2).
• Figure 8 is a bar graph showing relative NDP RNA expression levels (relative to mouse GAPDH (mGADPH)) in mouse explants transduced with Anc80.NDP.UTR (Construct 1) or Anc80.NDP (Construct 2).
• Figure 9 is an exemplary image of a Western blot showing secreted norrin (or
• Anc80.NDP.UTR (Construct 1) or Anc80.NDP (Construct 2).
• Figure 10 shows an exemplary image of EGFP protein expression in HEK cells using constructs described herein (mock, CAG-EGFP, NDP -EGFP).
• Figure 11 is an exemplary nucleic acid vector, Construct 6 (SEQ ID NO: 96,
• a 5TTR sequence (SEQ ID NO: 116), a CMV enhancer sequence (SEQ ID NO: 118), a CBA promoter sequence (SEQ ID NO: 119), a chimeric intron sequence (SEQ ID NO: 120), a IL2 stuffer sequence (SEQ ID NO: 122), a Hsp70 coding sequence (SEQ ID NO: 123), a bGH poly(A) sequence (SEQ ID NO: 125), and an ITR sequence (SEQ ID NO: 127).
• 5TTR sequence (SEQ ID NO: 116), a CMV enhancer sequence (SEQ ID NO: 118), a CBA promoter sequence (SEQ ID NO: 119), a chimeric intron sequence (SEQ ID NO: 120), a IL2 stuffer sequence (SEQ ID NO: 122), a Hsp70 coding sequence (SEQ ID NO: 123), a bGH poly(A) sequence (SEQ ID NO: 125), and an ITR sequence (
• Figure 12 is an exemplary nucleic acid vector, Construct 7 (SEQ ID NO: 97,
• 4179 bp that includes a 5’ITR sequence (SEQ ID NO: 128), a CMV enhancer sequence (SEQ ID NO: 130), a CBA promoter sequence (SEQ ID NO: 131), a chimeric intron sequence (SEQ ID NO: 132), a Hsp70 coding sequence (SEQ ID NO: 134), a bGH poly(A) sequence (SEQ ID NO: 136), and an ITR sequence (SEQ ID NO: 138).
• Figure 13 is an exemplary nucleic acid vector, Construct 8 (SEQ ID NO: 98,
• 4179 bp that includes a 5TTR sequence (SEQ ID NO: 139), a CMV enhancer sequence (SEQ ID NO: 141), a CBA promoter sequence (SEQ ID NO: 142), a chimeric intron sequence (SEQ ID NO: 143), a Hsp70 coding sequence (SEQ ID NO: 145), a 3xFLAG sequence (SEQ ID NO: 147), a T2A sequence (SEQ ID NO: 149), a tGFP sequence (SEQ ID NO: 150), a bGH poly (A) sequence (SEQ ID NO: 152), and an HR sequence (SEQ ID NO: 154).
• Figure 14 is an exemplary image of a Western blot showing secreted HSP70 protein detected in the lysate or supernatant of cochlea explant cultures transduced with CAG- Hsp70 (Construct 7), CAG-IL2ss-HSP70 (Construct 6), or CAG-Hsp70-3xFlag-tGFP (Construct 8).
• Figure 15 depicts a bar graph showing relative Hsp70 RNA expression levels
• Figure 16 are exemplary fluorescent images showing Myo7a staining following ex vivo cochlea tolerability of Anc80-CAG.IL2ss.HSP70 (left image: mock solution (control); right imageAnc80-CAG.IL2ss.HSP70 (Construct 6) (2.2E+10 vg/cochlea)).
• Figure 17 depicts a bar graph showing relative Hsp70 RNA expression levels
• Figure 18 is an exemplary image of a Western blot showing secreted HSP70 protein detected in supernatant (media) of HEK cells transduced with CAG-Hsp70 (Construct 7) (2.5E+5 MOI and 7.5E+5 MOI) and CAG-IL2ss-HSP70 (Construct 6) (2.4E+5 MOI and 7.2E+5 MOI).
• Figures 19A depicts a simplified wild-type AAV genome.
• Figure 19B depicts a simplified AAV constmct capable of expressing an NDP gene.
• Figure 19C depicts a simplified AAV construct capable of expressing an
• an ear can be described as including: an outer ear, middle ear, inner ear, hearing (acoustic) nerve, and auditory system (which processes sound as it travels from the ear to the brain).
• ears also help to maintain balance.
• disorders of the inner ear can cause hearing loss, tinnitus, vertigo, imbalance, or combinations thereof.
• Hearing loss can be the result of genetic factors, environmental factors, or a combination of genetic and environmental factors. About half of all people who have tinnitus- phantom noises in their auditory system (ringing, buzzing, chirping, humming, or beating)-also have an over-sensitivity to/reduced tolerance for certain sound frequency and volume ranges, known as hyperacusis (also spelled hyperacousis). A variety of non-syndromic and syndromic- related hearing losses will be known to those of skill in the art (e.g., DFNB4, and Pendred syndrome, respectively).
• Environmental causes of hearing impairment or loss may include, e.g., certain medications, specific infections before or after birth, and/or exposure to loud noise over an extended period.
• hearing loss can result from noise, ototoxic agents, presbycusis, disease, infection or cancers that affect specific parts of the ear.
• ischemic damage can cause hearing loss via pathophysiological mechanisms.
• intrinsic abnormalities like congenital mutations to genes that play an important role in cochlear anatomy or physiology, or genetic or anatomical changes in supporting and/or hair cells can be responsible for or contribute to hearing loss.
• Hearing loss and/or deafness is one of the most common human sensory deficits, and can occur for many reasons.
• a subject may be born with hearing loss or without hearing, while others may lose hearing slowly over time.
• Approximately 36 million American adults report some degree of hearing loss, and one in three people older than 60 and half of those older than 85 experience hearing loss.
• Approximately 1.5 in 1,000 children are born with profound hearing loss, and another two to three per 1,000 children are born with partial hearing loss (Smith et ah, 2005, Lancet 365:879-890, which is incorporated in its entirety herein by reference). More than half of these cases are attributed to a genetic basis (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499, which is incorporated in its entirety herein by reference).
• non-syndromic hearing loss and/or deafness is not associated with other signs and symptoms. In some embodiments, syndromic hearing loss and/or deafness occurs in conjunction with abnormalities in other parts of the body. Approximately 70 percent to 80 percent of genetic hearing loss and/or deafness cases are non-syndromic ;
• Non-syndromic deafness and/or hearing loss can have different patterns of inheritance, and can occur at any age.
• Types of non-syndromic deafness and/or hearing loss are generally named according to their inheritance patterns. For example, autosomal dominant forms are designated DFNA, autosomal recessive forms are DFNB, and X-linked forms are DFN. Each type is also numbered in the order in which it was first described. For example, DFNA1 was the first described autosomal dominant type of non-syndromic deafness.
• each parent of an individual with autosomal recessive hearing loss and/or deafness is a carrier of one copy of the mutated gene, but is not affected by this form of hearing loss.
• Another 20 percent to 25 percent of non-syndromic hearing loss and/or deafness cases are autosomal dominant, which means one copy of the altered gene in each cell is sufficient to result in deafness and/or hearing loss.
• X-linked pattern of inheritance which means the mutated gene responsible for the condition is located on the X chromosome (one of the two sex chromosomes).
• Males with X-linked non-syndromic hearing loss and/or deafness tend to develop more severe hearing loss earlier in life than females who inherit a copy of the same gene mutation.
• a characteristic of X-linked inheritance is that fathers cannot pass X-linked traits to their sons.
• Mitochondrial non- syndromic deafness which results from changes to mitochondrial DNA, occurs in less than one percent of cases in the United States.
• the altered mitochondrial DNA is passed from a mother to all of her sons and daughters. This type of deafness is not inherited from fathers.
• the causes of syndromic and non-syndromic deafness and/or hearing loss are complex.
• researchers have identified more than 30 genes that, when altered, are associated with syndromic and/or non- syndromic deafness and/or hearing loss; however, some of these genes have not been fully characterized. Different mutations in the same gene can be associated with different types of deafness and/or hearing loss, and some genes are associated with both syndromic and non- syndromic deafness and/or hearing loss.
• deafness and/or hearing loss can be conductive (arising from the ear canal or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed.
• non-syndromic deafness and/or hearing loss is associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural deafness).
• sensorineural hearing loss can be due to poor hair cell function.
• sensorineural hearing impairments involve the eighth cranial nerve (the vestibulocochlear nerve) or the auditory portions of the brain. In some such embodiments, only the auditory centers of the brain are affected.
• cortical deafness may occur, where sounds may be heard at normal thresholds, but quality of sound perceived is so poor that speech cannot be understood.
• Hearing loss that results from changes in the middle ear is called conductive hearing loss.
• Some forms of non-syndromic deafness and/or hearing loss involve changes in both the inner ear and the middle ear, called mixed hearing loss.
• Hearing loss and/or deafness that is present before a child learns to speak can be classified as prelingual or congenital.
• Hearing loss and/or deafness that occurs after the development of speech can be classified as postlingual.
• Most autosomal recessive loci related to syndromic or non-syndromic hearing loss cause prelingual severe-to-profound hearing loss.
• hair cells are sensory receptors for both auditory and vestibular systems of vertebrate ears. Hair cells detect movement in the
• hair cells are located within the cochlea of the ear, in the organ of Corti.
• Mammalian ears are known to have two types of hair cells - inner hair cells and outer hair cells.
• Outer hair cells can amplify low level sound frequencies, either through mechanical movement of hair cell bundles or electrically-driven movement of hair cell soma.
• Inner hair cells transform vibrations in cochlear fluid into electrical signals that the auditory nerve transmits to the brain.
• hair cells may be abnormal at birth, or damaged during the lifetime of an individual.
• outer hair cells may be able to regenerate.
• inner hair cells are not capable of regeneration after illness or injury.
• sensorineural hearing loss is due to abnormalities in hair cells.
• supporting cells may fulfil numerous functions, and include a number of cell types, including but not limited to Hensen’s cells, Deiters’ cells, pillar cells, Claudius cells, inner phalangeal cells, and border cells.
• sensorineural hearing loss is due to abnormalities in supporting cells.
• supporting cells may be abnormal at birth, or damaged during the lifetime of an individual.
• supporting cells may be able to regenerate. In some embodiments, certain supporting cells may not be capable of regeneration.
• mutations in an NDP gene that encodes the“norrin cysteine knot growth factor” (NDP) protein may cause hearing loss and vision loss.
• mutations in NDP lead to Nome disease pseudoglioma.
• composition including a single adeno-associated virus (AAV) vector, wherein the single AAV vector that includes a nucleic acid sequence that encodes a secreted target protein; and when introduced into a primate cell, a nucleic acid encoding a full- length secreted target protein is generated at the locus of the secreted target protein, and the primate cell expresses and secretes the secreted target protein.
• AAV adeno-associated virus
• compositions including at least two different nucleic acid vectors, wherein: each of the at least two different vectors includes a coding sequence that encodes a different portion of a secreted target protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes a full-length secreted target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the secreted target protein genomic DNA, and lacks an intronic sequence between the two neighboring exons; and when introduced into a mammalian cell the at least two different vectors undergo concatamerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length secreted target protein.
• compositions including two different nucleic acid vectors, wherein: a first nucleic acid vector of the two different nucleic acid vectors includes a promoter, a first coding sequence that encodes an N-terminal portion of an secreted target protein positioned 3’ of the promoter, and a splicing donor signal sequence positioned at the 3’ end of the first coding sequence; and a second nucleic acid vector of the two different nucleic acid vectors includes a splicing acceptor signal sequence, a second coding sequence that encodes a C- terminal portion of an secreted target protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the amino acid sequences of the encoded portions do not overlap, wherein no single vector of the two different vectors encodes a full-length secreted target protein, and
• compositions including: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of an secreted target protein positioned 3’ of the promoter, a splicing donor signal sequence positioned at the 3’ end of the first coding sequence, and a first detectable marker gene positioned 3’ of the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second detectable marker gene, a splicing acceptor signal sequence positioned 3’ of the second detectable marker gene, a second coding sequence that encodes a C-terminal portion of an secreted target protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the respective amino acid sequences of the encoded portions do not overlap with each
• compositions including: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of an secreted target protein positioned 3’ to the promoter, a splicing donor signal sequence positioned at the 3’ end of the first coding sequence, and a FI phage recombinogenic region positioned 3’ to the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second FI phage recombinogenic region, a splicing acceptor signal sequence positioned 3’ of the second FI phage recombinogenic region, a second coding sequence that encodes a C-terminal portion of an secreted target protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in
• Also provided herein are methods including introducing into a cochlea of a mammal a therapeutically effective amount of any of the compositions described herein.
• Also provided herein are methods of treating or preventing vision loss in a subject identified as having a defective NDP gene that include: administering a therapeutically effective amount of any of the compositions described herein into an inner ear or central nervous system of the subject, or systemically administering a therapeutically effective amount of any of the compositions described herein to the subject.
• compositions and kits that include any of the compositions or AAV vectors described herein.
• compositions, kits and methods are described herein and can be used in any combination without limitation.
• secreted target protein refers to a protein encoded by DNA that if expressed in a cell (e.g., any of the exemplary cells described herein) can be secreted.
• secreted target protein also refers to a protein that includes a secretion signal.
• secreted target protein is a NDP protein (e.g., any of the NDP proteins described herein).
• the secreted target protein is an HSPA1 A protein (e.g., any of the HPSA1A proteins described herein).
• the term“secreted target protein” means a protein that is expressed and secreted by a cell in a primate, and functionally contributes, at least in part, to the hearing in the primate.
• secreted target proteins include NDP, HSPA1A, DNAJB 1, or DNAJB5.
• mutation in a secreted target gene refers to a modification in a wildtype secreted target gene that results in the production of a secreted target protein having one or more of: a deletion in one or more amino acids, one or more amino acid substitutions, and one or more amino acid insertions as compared to the wildtype secreted target protein, and/or results in a decrease in the expressed level of the encoded secreted target protein in a primate cell as compared to the expressed level of the encoded secreted target protein in a primate cell not having a mutation.
• a mutation can result in the production of a secreted target protein having a deletion in one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids). In some embodiments, the mutation can result in a frame shift in the secreted target gene.
• amino acids e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
• NDP Norrin Cystine Knot Growth Factor
• NDP “norrin cystine knot growth factor”
• ND gene is located on chromosome Xpl 1.3. It contains 3 exons, encompassing ⁇ kilobases (kb) (NCBI Accession No. NG_009832.1). NDP encodes secreted norrin protein that plays a role in retina vascularization of Wnt signaling pathway through (FZDA) and (LRP5) coreceptor.
• NDP neurodegenerative disease
• Norrie Disease Pseudoglioma an X-linked recessive syndromic hearing loss that is characterized by early childhood retinopathy. About one third of individuals with Norrie disease develop progressive hearing loss, and more than half experience developmental delays in motor skills.
• Various mutations in the NDP gene have been associated with hearing loss (e.g.,
• Nome Disease Pseudoglioma the p.His4ArgfsX21, p.Asp23GlufsX9, p.Arg38Cys, p.Ile48ValfsX55, p.His50Asp, p.Ser57*, p.Cys93Arg, p.Lysl04Gln, p.Glyl l3Asp, p.Argl21Gln, and p.Cysl26Arg mutation have each been associated with Nome disease pseudoglioma. See, e.g., Musada et al., Mol. Vis.
• active NDP protein means a protein encoded by DNA that, if substituted for both wildtype alleles encoding full-length NDP protein in auditory hair cells, or ocular cells, of what is otherwise a wildtype mammal, and if expressed in the auditory hair cells, or ocular cells, of that mammal, results in that mammal’s having a level of hearing, or vision, approximating the normal level of hearing, or vision, of a similar mammal that is entirely wildtype.
• active NDP proteins are full-length NDP proteins (e.g., any of the full-length NDP proteins described herein).
• an active NDP protein can include a sequence of a wildtype, full- length NDP protein (e.g., a wildtype, human, full-length NDP protein) including about 1 to about 24 amino acid substitutions (e.g., about 1 to about 22 amino acid substitutions, about 1 to about 20 amino acid substitutions, about 1 to about 18 amino acid substitutions, about 1 to about 16 amino acid substitutions, about 1 to about 15 amino acid substitutions, about 1 to about 14 amino acid substitutions, about 1 to about 12 amino acid substitutions, about 1 to about 10 amino acid substitutions, about 1 to about 8 amino acid substitutions, about 1 to about 6 amino acid substitutions, about 1 to about 5 amino acid substitutions, about 1 to about 4 amino acid substitutions, about 1 to about 2 amino acid substitutions, about 2 to about 24 amino acid substitutions, about 2 to about 22 amino acid substitutions, about 2 to about 20 amino acid substitutions, about 2 to about 18 amino acid substitutions, about 2 to about 16 amino acid substitutions, about 2 to about 15 amino acid substitutions,
• amino acids that are not conserved between wildtype NDP proteins from different species can be mutated without losing activity, while those amino acids that are conserved between wildtype NDP proteins from different species should not be mutated as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.
• An active NDP protein can include, e.g., a sequence of a wildtype, full-length
• NDP protein (e.g., a wildtype, human, full-length NDP protein) that has about 1 to about 80 amino acids (e.g., about 1 to about 75 amino acids, about 1 to about 70 amino acids, about 1 to about 65 amino acids, about 1 to about 60 amino acids, about 1 to about 55 amino acids, about 1 to about 50 amino acids, about 1 to about 45 amino acids, about 1 to about 40 amino acids, about 1 to about 35 amino acids, about 1 to about 30 amino acids, about 1 to about 25 amino acids, about 1 to about 20 amino acids, about 1 to about 15 amino acids, about 1 to about 10 amino acids, about 1 to about 5 amino acids, about 5 to about 80 amino acids, about 5 to about 75 amino acids, about 5 to about 70 amino acids, about 5 to about 65 amino acids, about 5 to about 60 amino acids, about 5 to about 55 amino acids, about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids,
• an active NDP protein can, e.g., include the sequence of a wildtype, full-length NDP protein where 1 amino acid to 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 amino acids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 5 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 3 amino acids, about 2 amino acids to 50 amino acids, about 2 amino acids to 45 amino acids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35 amino acids, about 2 amino acids to 30 amino acids, about 2 amino acids to 25 amino acids, about 2 amino acids to 20 amino acids, about 2 amino acids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2 amino acids to 9 amino acids.
• the 1 amino acid to 50 amino acids can be inserted as a contiguous sequence into the sequence of a wildtype, full-length NDP protein. In some examples, the 1 amino acid to 50 amino acids (or any subrange thereof) are inserted in multiple, non-contiguous places in the sequence of a wildtype, full-length NDP protein. As can be appreciated in the art, the 1 amino acid to 50 amino acids can be inserted into a portion of the sequence of a wildtype, full-length NDP protein that is not well- conserved between species.
• Methods of detecting mutations in a gene are well-known in the art.
• Non-limiting examples of such techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.
• An exemplary human wildtype NDP protein is or includes the sequence of SEQ
• Non-limiting examples of nucleic acid encoding a wildtype NDP protein are or include SEQ D NO: 2 or SEQ ID NO: 34.
• SEQ D NO: 2 or SEQ ID NO: 34 At least some or all of the codons in SEQ ID NO: 2 or SEQ ID NO: 34 can be codon-optimized to allow for optimal expression in a non-human mammal.
• Exemplary wildtype NDP protein sequences are or include SEQ ID NO: 1, 33, 35,
• polynucleotides e.g., polynucleotides comprising an NDP gene or characteristic portion thereof, as well as
• compositions including such polynucleotides and methods utilizing such polynucleotides and/or compositions.
• a polynucleotide comprising an NDP gene or characteristic portion thereof can be DNA or RNA.
• DNA can be genomic DNA or cDNA.
• RNA can be an mRNA.
• a polynucleotide comprises exons and/or introns of an NDP gene.
• a gene product is expressed from a polynucleotide comprising an NDP gene or characteristic portion thereof.
• expression of such a polynucleotide can utilize one or more control elements (e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.).
• a polynucleotide provided herein can include one or more control elements.
• an NDP gene is a mammalian NDP gene. In some embodiments, an NDP gene is a murine NDP gene. In some embodiments, an NDP gene is a primate NDP gene. In some embodiments, a NDP gene is a human NDP gene.
• An exemplary human NDP cDNA sequence is or includes the sequence of SEQ ID NO: 57.
• An exemplary human NDP genomic DNA sequence can be found in SEQ ID NO: 79.
• An exemplary human NDP cDNA sequence including untranslated regions is or includes the sequence of SEQ ID NO: 114.
• a non-limiting example of a human wildtype NDP genomic DNA sequence is
• SEQ ID NO: 79 The exons in SEQ ID NO: 79 are: nucleotide positions 1-87 (exon 1);
• nucleotide positions 88-468 (exon 2) and nucleotide positions 469-1719 (exon 3).
• AAAAC AGAC AG T TCAT T TCTGAT GGAC T C T C AAC G C T G C AT GAGAC AC CAT TAT GTCGAT TCTA TCAGTCACCCACTGTACAAATGTAGCTCAAAGATGGTGCTCCTGGCCAGATGTGAGGGGCACTG CAGCCAGGCATCACGCTCTGAGCCCT TGGTGTCCTTCAGCACTGTCCTCAAGCAACCTT TCCGT TCCTCCTGTCACTGCTGCCGACCCCAGACTTCCAAGCTGAAGGCTCTGCGTCTGCGCTGCTCAG GGGGCATGCGACTTACTGCCACT TACCGGTACATCCTCTCCTGTCACTGTGAGGAATGCAGCTC C
• an NDP gene is a mammalian NDP gene. In some embodiments, an NDP gene is a murine NDP gene. In some embodiments, an NDP gene is a primate NDP gene. In some embodiments, a NDP gene is a human NDP gene.
• a polypeptide comprises a NDP protein or characteristic portion thereof.
• a NDP protein or characteristic portion thereof is mammalian NDP protein or characteristic portion thereof, e.g., primate NDP protein or characteristic portion thereof
• a NDP protein or characteristic portion thereof is a human NDP protein or characteristic portion thereof.
• a polypeptide provided herein comprises post-translational modifications.
• a NDP protein or characteristic portion thereof provided herein comprises post-translational modifications.
• post-translational modifications can comprise but is not limited to glycosylation (e.g., N-linked glycosylation, O- linked glycosylation), phosphorylation, acetylation, amidation, hydroxylation, methylation, ubiquitylation, sulfation, and/or a combination thereof.
• An exemplary human NDP protein sequence is or includes the sequence of SEQ
• An exemplary human NDP protein sequence with a c-terminal flag tag is or includes the sequence of SEQ ID NO: 94.
• As exemplary mouse NDP protein sequence is or includes the sequence of SEQ ID NO: 80.
• As exemplary rhesus monkey NDP protein sequence is or includes the sequence of SEQ ID NO: 82.
• As exemplary rat NDP protein sequence is or includes the sequence of SEQ ID NO: 83.
• As exemplary chimpanzee NDP protein sequence is or includes the sequence of SEQ ID NO: 84.
• Exemplary Human Mature NDP Protein (SEQ ID NO: 56) (NCBI Accession No.
• a secreted target protein is a heat shock protein.
• a heat shock protein is a heat shock protein family A (Hsp70) member 1A (HSPA1A).
• a heat shock protein is a heat shock protein 40 (Hsp40)/DNJ family member, e.g., Hsp40.
• HSPA1A Heat Shock Protein Family A
• HSPA1A Heat Shock Protein Family A
• HSPA1A The human HSPA1A gene is located on chromosome 6p21.33. It contains 1 exon, encompassing ⁇ 2400 bp (NCBI Accession No. NC_000006.12). HSPA1 A encodes a 70kDa heat shock protein, which stabilizes proteins against protein aggregation and is involved in protein folding.
• Hair cells are susceptible to death when exposed to therapeutic drugs (such as aminoglycoside antibiotics or cisplatin) with ototoxic side effects.
• therapeutic drugs such as aminoglycoside antibiotics or cisplatin
• HSP70 heat shock protein 70
• HO-1 heme oxygenase- 1
• HSP32 heme oxygenase- 1
• adenovirus-mediated infection of inner ear supporting cells with HSP70 was shown to inhibit hair cell death (see, e.g., Lindsay May et al J Clin Invest.
• polymorphisms in Hsp70 may render the cochlea susceptible to hearing loss (such as a polymorphism described by Konings et al.,“Variations in HSP70 genes associated with noise-induced hearing loss in two independent populations”, Eur J Hum Genet. 2009 Mar; 17(3): 329-33, the contents of which is hereby incorporated by reference in its entirety).
• active HSPA1A protein means a protein encoded by
• active HSPA1A proteins are full-length HSPA1A proteins (e.g., any of the full-length HSPA1A proteins described herein).
• an active HSPA1 A protein can include a sequence of a wildtype, full-length HSPA1 A protein (e.g., a wildtype, human, full-length HSPA1 A protein) including about 1 to about 200 amino acid substitutions (e.g., about 1 to 190 amino acid substitutions, about 1 to about 180 amino acid substitutions, about 1 to about 160 amino acid substitutions, about 1 to about 150 amino acid substitutions, about 1 to about 140 amino acid substitutions, about 1 to about 130 amino acid substitutions, about 1 to about 120 amino acid substitutions, about 1 to about 110 amino acid substitutions, about 1 to about 100 amino acid substitutions, about 1 to about 90 amino acid substitutions, about 1 to about 80 amino acid substitutions, about 1 to about 70 amino acid substitutions, about 1 to about 60 amino acid substitutions, about 1 to about 50 amino acid substitutions, about 1 to about 40 amino acid substitutions, about 1 to about 30 amino acid substitutions, about 1 to about 25 amino acid substitutions, about 1 to about 20 amino acid substitutions, about 1 to about
• substitutions about 140 to about 150 amino acid substitutions, about 150 to about 200 amino acid substitutions, about 150 to about 180 amino acid substitutions, about 150 to about 160 amino acid substitutions, about 160 to about 200 amino acid substitutions, about 160 to about 180 amino acid substitutions, or about 180 to about 200 amino acid substitutions).
• An active HSPA1 A protein can include, e.g., a sequence of a wildtype, full-length
• HSPA1 A protein (e.g., a wildtype, human, full-length HSPA1 A protein) that has about 1 to about 100 amino acids (e.g., about 1 to about 95 amino acids, about 1 to about 90 amino acids, about 1 to about 85 amino acids, about 1 to about 80 amino acids, about 1 to about 75 amino acids, about 1 to about 70 amino acids, about 1 to about 65 amino acids, about 1 to about 60 amino acids, about 1 to about 55 amino acids, about 1 to about 50 amino acids, about 1 to about 45 amino acids, about 1 to about 40 amino acids, about 1 to about 35 amino acids, about 1 to about 30 amino acids, about 1 to about 25 amino acids, about 1 to about 20 amino acids, about 1 to about 15 amino acids, about 1 to about 10 amino acids, about 1 to about 5 amino acids, about 5 to about 100 amino acids, about 5 to about 95 amino acids, about 5 to about 90 amino acids, about 5 to about 85 amino acids, about 5 to about 80 amino acids, about 5 to about 75 amino acids, about 5 to about 70 amino acids, about 5 to about
• an active HSPA1 A protein can, e.g., include the sequence of a wildtype, full-length HSPA1 A protein where 1 amino acid to 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 amino acids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 5 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 3 amino acids, about 2 amino acids to 50 amino acids, about 2 amino acids to 45 amino acids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35 amino acids, about 2 amino acids to 30 amino acids, about 2 amino acids to 25 amino acids, about 2 amino acids to 20 amino acids, about 2 amino acids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2 amino acids
• the 1 amino acid to 50 amino acids can be inserted as a contiguous sequence into the sequence of a wildtype, full-length HSPA1A protein. In some examples, the 1 amino acid to 50 amino acids (or any subrange thereof) are inserted in multiple, non-contiguous places in the sequence of a wildtype, full-length HSPA1A protein. As can be appreciated in the art, the 1 amino acid to 50 amino acids can be inserted into a portion of the sequence of a wildtype, full-length HSPA1 A protein that is not well-conserved between species.
• Methods of detecting mutations in a gene are well-known in the art.
• Non-limiting examples of such techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.
• Exemplary wildtype HSPA1 A protein sequences are or include SEQ ID NO: 85,
• the present disclosure provides polynucleotides, e.g., polynucleotides comprising an HSPA1A gene or characteristic portion thereof, as well as compositions including such polynucleotides and methods utilizing such polynucleotides and/or compositions.
• a polynucleotide comprising an HSPA1A gene or characteristic portion thereof can be DNA or RNA.
• DNA can be genomic DNA or cDNA.
• RNA can be an mRNA.
• a polynucleotide comprises exons and/or introns of an HSPA1 A gene.
• a gene product is expressed from a polynucleotide comprising an HSPA1 A gene or characteristic portion thereof.
• expression of such a polynucleotide can utilize one or more control elements (e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.).
• control elements e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.
• a polynucleotide provided herein can include one or more control elements.
• an HSPA1A gene is a mammalian HSPA1A gene. In some embodiments, an HSPA1A gene is a murine HSPA1A gene. In some embodiments, an HSPA1 A gene is a primate HSPA1 A gene. In some embodiments, a HSPA1 A gene is a human HSPA1 A gene.
• An exemplary human HSPA1 A cDNA sequence is or includes the sequence of SEQ ID NO: 86.
• An exemplary human HSPA1A genomic DNA sequence can be found in SEQ ID NO: 40.
• An exemplary human HSPA1A cDNA sequence including untranslated regions is or includes the sequence of SEQ ID NO: 115.
• Exemplary Mature HSPA1A cDNA including untranslated regions (SEQ ID NO: 115)
• a non-limiting example of a human wildtype HSPA1A genomic DNA sequence is SEQIDNO: 87.
• an HSPA1A gene is a mammalian HSPA1 A gene. In some embodiments, an HSPA1 A gene is a murine HSPA1 A gene. In some embodiments, an HSPA1A gene is a primate HSPA1A gene. In some embodiments, a HSPA1 A gene is a human HSPA1 A gene.
• a polypeptide comprises a HSPA1A protein or characteristic portion thereof.
• a HSPA1A protein or characteristic portion thereof is mammalian HSPA1 A protein or characteristic portion thereof, e.g., primate HSPA1 A protein or characteristic portion thereof.
• a HSPA1A protein or characteristic portion thereof is a human HSPA1 A protein or characteristic portion thereof.
• a polypeptide provided herein comprises post-translational modifications.
• a HSPA1A protein or characteristic portion thereof provided herein comprises post-translational modifications.
• post- translational modifications can comprise but is not limited to glycosylation (e g., N-linked glycosylation, O-linked glycosylation), phosphorylation, acetylation, amidation, hydroxyl ati on, methylation, ubiquitylation, sulfation, and/or a combination thereof.
• An exemplary human HSPA1A protein sequence is or includes the sequence of
• SEQ ID NO: 85 An exemplary human HSPA1 A protein sequence with a c-terminal flag tag is or includes the sequence of SEQ ID NO: 95.
• exemplary mouse HSPA1A protein sequence is or includes the sequence of SEQ ID NO: 88.
• exemplary rhesus monkey HSPA1A protein sequence is or includes the sequence of SEQ ID NO: 90.
• exemplary rat HSPA1 A protein sequence is or includes the sequence of SEQ ID NO: 91.
• exemplary cattle HSPA1A protein sequence is or includes the sequence of SEQ ID NO: 92.
• Exemplary Human Mature HSPA1A Protein (SEQ ID NO: 85) (NCBI Accession No.
• Exemplary Cattle HSPA1A Protein (SEQ ID NO: 92) (NCBI Accession No. NP 976067.3)
• a secreted protein described herein can be a heat shock protein from the
• Hsp40/DNAJ family Proteins in the Hsp40/DNAJ family comprise a 70 amino-acid consensus sequence known as the J domain, which interacts with a HSP70 protein. Without being bound to any particular theory, it is believed that proteins in the Hsp40/DNAJ family play a role in regulating the adenosine triphosphatase (ATPase) activity of HSP70 heat-shock proteins (e.g., HSPA1A).
• ATPase adenosine triphosphatase
• a protein in the Hsp40/DNAJ family is encoded by a Type
• a protein in the Hsp40/DNAJ family is encoded by a Type 2 (subfamily B) gene.
• a protein in the Hsp40/DNAJ family is encoded by a Type 3 (subfamily C) gene.
• a protein in the Hsp40/DNAJ family is encoded by a J-like gene.
• a protein in the Hsp40/DNAJ family is encoded by a gene listed in Table 1. Exemplary sequences for the genes can be found by reference to the Gene ID in Table 1. In some embodiments, a protein in the Hsp40/DNAJ family is listed in Table 1. Exemplary sequences for the proteins can be found by reference to the UniProt ID in Table 1.
• an Hsp40 protein is encoded by a DNAJB1 gene.
• the human DNAJB1 gene is located on chromosome Chrl9: 14,514,770-14,529,770. It contains 7 exons (NCBI Accession No. NC_000019.10).
• DNAJB1 encodes a 40kDa heat shock protein.
• active DNAJB1 protein means a protein encoded by
• active DNAJB1 proteins are full-length DNAJB1 proteins (e.g., any of the full-length DNAJB1 proteins described herein).
• an active DNAJB1 protein can include a sequence of a wildtype, full-length DNAJB1 protein (e.g., a wildtype, human, full-length DNAJB1 protein) including about 1 to about 200 amino acid substitutions (e.g., about 1 to 190 amino acid substitutions, about 1 to about 180 amino acid substitutions, about 1 to about 160 amino acid substitutions, about 1 to about 150 amino acid substitutions, about 1 to about 140 amino acid substitutions, about 1 to about 130 amino acid substitutions, about 1 to about 120 amino acid substitutions, about 1 to about 110 amino acid substitutions, about 1 to about 100 amino acid substitutions, about 1 to about 90 amino acid substitutions, about 1 to about 80 amino acid substitutions, about 1 to about 70 amino acid substitutions, about 1 to about 60 amino acid substitutions, about 1 to about 50 amino acid substitutions, about 1 to about 40 amino acid substitutions, about 1 to about 30 amino acid substitutions, about 1 to about 25 amino acid substitutions, about 1 to about 20 amino acid substitutions, about 1 to about
• substitutions about 140 to about 150 amino acid substitutions, about 150 to about 200 amino acid substitutions, about 150 to about 180 amino acid substitutions, about 150 to about 160 amino acid substitutions, about 160 to about 200 amino acid substitutions, about 160 to about 180 amino acid substitutions, or about 180 to about 200 amino acid substitutions).
• amino acids that are not conserved between wildtype DNAJB 1 proteins from different species can be mutated without losing activity, while those amino acids that are conserved between wildtype DNAJB 1 proteins from different species should not be mutated as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.
• An active DNAJB1 protein can include, e.g., a sequence of a wildtype, full- length DNAJB 1 protein (e.g., a wildtype, human, full-length DNAJB1 protein) that has about 1 to about 100 amino acids (e.g., about 1 to about 95 amino acids, about 1 to about 90 amino acids, about 1 to about 85 amino acids, about 1 to about 80 amino acids, about 1 to about 75 amino acids, about 1 to about 70 amino acids, about 1 to about 65 amino acids, about 1 to about 60 amino acids, about 1 to about 55 amino acids, about 1 to about 50 amino acids, about 1 to about 45 amino acids, about 1 to about 40 amino acids, about 1 to about 35 amino acids, about 1 to about 30 amino acids, about 1 to about 25 amino acids, about 1 to about 20 amino acids, about 1 to about 15 amino acids, about 1 to about 10 amino acids, about 1 to about 5 amino acids, about 5 to about 100 amino acids, about 5 to about 95 amino acids, about 5 to about 90 amino acids, about 5 to about 85
• an active DNAJB1 protein can, e.g., include the sequence of a wildtype, full-length DNAJB 1 protein where 1 amino acid to 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 amino acids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to
• amino acids to 4 amino acids 1 amino acid to 3 amino acids, about 2 amino acids to 50 amino acids, about 2 amino acids to 45 amino acids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35 amino acids, about 2 amino acids to 30 amino acids, about 2 amino acids to 25 amino acids, about 2 amino acids to 20 amino acids, about 2 amino acids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2 amino acids to 9 amino acids, about 2 amino acids to 8 amino acids, about 2 amino acids to 7 amino acids, about 2 amino acids to 6 amino acids, about 2 amino acids to 5 amino acids, about 2 amino acids to 4 amino acids, about 3 amino acids to 50 amino acids, about 3 amino acids to 45 amino acids, about 3 amino acids to 40 amino acids, about 3 amino acids to 35 amino acids, about 3 amino acids to 30 amino acids, about 3 amino acids to 25 amino acids, about 3 amino acids to 20 amino acids, about 3 amino acids to 15 amino acids, about 3 amino acids to 10 amino acids, about 3 amino acids to 9 amino acids, about 3 amino acids to 8 amino acids, about
• the 1 amino acid to 50 amino acids can be inserted as a contiguous sequence into the sequence of a wildtype, full-length DNAJB1 protein. In some examples, the 1 amino acid to 50 amino acids (or any subrange thereof) are inserted in multiple, non-contiguous places in the sequence of a wildtype, full-length DNAJB1 protein. As can be appreciated in the art, the 1 amino acid to 50 amino acids can be inserted into a portion of the sequence of a wildtype, full-length DNAJB1 protein that is not well-conserved between species.
• Methods of detecting mutations in a gene are well-known in the art. Non-limiting examples of such techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.
• RT-PCR real-time polymerase chain reaction
• PCR PCR
• sequencing Southern blotting
• Northern blotting exemplary wildtype DNAJB1 protein sequences are or include SEQ ID NO: 38,
• the present disclosure provides polynucleotides, e.g., polynucleotides comprising an DNAJB1 gene or characteristic portion thereof, as well as compositions including such polynucleotides and methods utilizing such polynucleotides and/or compositions.
• a polynucleotide comprising an DNAJB1 gene or characteristic portion thereof can be DNA or RNA.
• DNA can be genomic DNA or cDNA.
• RNA can be an mRNA.
• a polynucleotide comprises exons and/or introns of an DNAJB1 gene.
• a gene product is expressed from a polynucleotide comprising an DNAJB1 gene or characteristic portion thereof.
• expression of such a polynucleotide can utilize one or more control elements (e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.).
• control elements e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.
• a polynucleotide provided herein can include one or more control elements.
• an DNAJB1 gene is a mammalian DNAJB 1 gene. In some embodiments, an DNAJB1 gene is a murine DNAJB1 gene. In some embodiments, an DNAJB1 gene is a primate DNAJB 1 gene. In some embodiments, a DNAJB1 gene is a human DNAJB1 gene.
• An exemplary human DNAJB1 genomic sequence is or includes SEQ ID NO:
• An exemplary human DNAJB1 cDNA sequence is or includes the sequence of SEQ ID NO: 156 orl58.
• An exemplary human DNAJB1 cDNA sequence including untranslated regions is or includes the sequence of SEQ ID NO: 113, 157, or 159.
• Exemplary Human DNAJB1 cDNA including untranslated regions Variant 3 (SEQ ID NO: 159)
• Exemplary Human Mature DNAJB1 Protein Isoform 2 (SEQ ID NO: 161) MFAEFFGGRNPFDTFFGQRNGEEGMDIDDPFSGFPMGMGGFTNVNFGRSRSAQEPARKKQDPPV THDLRVSLEEIYSGCTKKMKISHKRLNPDGKSIRNEDKILTIEVKKG KEGTKITFPKEGDQTS NNIPADIVFVLKDKPHNIFKRDGSDVIYPARISLREALCGCTVNVPTLDGRTIPWFKDVIRPG MRRKVPGEGLPLPKTPEKRGDLI IEFEVIFPERIPQTSRTVLEQVLPI
• Heat Shock Protein 40 aJ homolog subfamily B member 5 (DNAJB5)
• an Hsp40 protein is encoded by a DNAJB5 gene.
• the human DNAJB5 gene is located on chromosome Chrl9: 14,514,770-14,529,770. It contains 7 exons (NCBI Accession No. NC_000019.10).
• DNAJB5 encodes a 40k Da heat shock protein.
• active DNAJB5 protein means a protein encoded by
• active DNAJB5 proteins are full-length DNAJB5 proteins (e.g., any of the full-length DNAJB5 proteins described herein).
• an active DNAJB5 protein can include a sequence of a wildtype, full-length DNAJB5 protein (e.g., a wildtype, human, full-length DNAJB5 protein) including about 1 to about 200 amino acid substitutions (e.g., about 1 to 190 amino acid substitutions, about 1 to about 180 amino acid substitutions, about 1 to about 160 amino acid substitutions, about 1 to about 150 amino acid substitutions, about 1 to about 140 amino acid substitutions, about 1 to about 130 amino acid substitutions, about 1 to about 120 amino acid substitutions, about 1 to about 110 amino acid substitutions, about 1 to about 100 amino acid substitutions, about 1 to about 90 amino acid substitutions, about 1 to about 80 amino acid substitutions, about 1 to about 70 amino acid substitutions, about 1 to about 60 amino acid substitutions, about 1 to about 50 amino acid substitutions, about 1 to about 40 amino acid substitutions, about 1 to about 30 amino acid substitutions, about 1 to about 25 amino acid substitutions, about 1 to about 20 amino acid substitutions, about 1 to about
• substitutions about 140 to about 150 amino acid substitutions, about 150 to about 200 amino acid substitutions, about 150 to about 180 amino acid substitutions, about 150 to about 160 amino acid substitutions, about 160 to about 200 amino acid substitutions, about 160 to about 180 amino acid substitutions, or about 180 to about 200 amino acid substitutions).
• amino acids that are not conserved between wildtype DNAJB5 proteins from different species can be mutated without losing activity, while those amino acids that are conserved between wildtype DNAJB5 proteins from different species should not be mutated as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.
• An active DNAJB5 protein can include, e.g., a sequence of a wildtype, full- length DNAJB5 protein (e.g., a wildtype, human, full-length DNAJB5 protein) that has about 1 to about 100 amino acids (e.g., about 1 to about 95 amino acids, about 1 to about 90 amino acids, about 1 to about 85 amino acids, about 1 to about 80 amino acids, about 1 to about 75 amino acids, about 1 to about 70 amino acids, about 1 to about 65 amino acids, about 1 to about 60 amino acids, about 1 to about 55 amino acids, about 1 to about 50 amino acids, about 1 to about 45 amino acids, about 1 to about 40 amino acids, about 1 to about 35 amino acids, about 1 to about 30 amino acids, about 1 to about 25 amino acids, about 1 to about 20 amino acids, about 1 to about 15 amino acids, about 1 to about 10 amino acids, about 1 to about 5 amino acids, about 5 to about 100 amino acids, about 5 to about 95 amino acids, about 5 to about 90 amino acids, about 5 to about 85
• an active DNAJB5 protein can, e.g., include the sequence of a wildtype, full-length DNAJB5 protein where 1 amino acid to 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 amino acids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to
• amino acids to 4 amino acids 1 amino acid to 3 amino acids, about 2 amino acids to 50 amino acids, about 2 amino acids to 45 amino acids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35 amino acids, about 2 amino acids to 30 amino acids, about 2 amino acids to 25 amino acids, about 2 amino acids to 20 amino acids, about 2 amino acids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2 amino acids to 9 amino acids, about 2 amino acids to 8 amino acids, about 2 amino acids to 7 amino acids, about 2 amino acids to 6 amino acids, about 2 amino acids to 5 amino acids, about 2 amino acids to 4 amino acids, about 3 amino acids to 50 amino acids, about 3 amino acids to 45 amino acids, about 3 amino acids to 40 amino acids, about 3 amino acids to 35 amino acids, about 3 amino acids to 30 amino acids, about 3 amino acids to 25 amino acids, about 3 amino acids to 20 amino acids, about 3 amino acids to 15 amino acids, about 3 amino acids to 10 amino acids, about 3 amino acids to 9 amino acids, about 3 amino acids to 8 amino acids, about
• the 1 amino acid to 50 amino acids can be inserted as a contiguous sequence into the sequence of a wildtype, full-length DNAJB5 protein. In some examples, the 1 amino acid to 50 amino acids (or any subrange thereof) are inserted in multiple, non-contiguous places in the sequence of a wildtype, full-length DNAJB5 protein. As can be appreciated in the art, the 1 amino acid to 50 amino acids can be inserted into a portion of the sequence of a wildtype, full-length DNAJB5 protein that is not well-conserved between species.
• Methods of detecting mutations in a gene are well-known in the art.
• Non-limiting examples of such techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.
• Exemplary wildtype DNAJB5 protein sequences are or include SEQ ID NO: 38,
• the present disclosure provides polynucleotides, e.g., polynucleotides comprising an DnaJ homolog subfamily B member 5 (DNAJB5) gene or characteristic portion thereof, as well as compositions including such polynucleotides and methods utilizing such polynucleotides and/or compositions.
• polynucleotides e.g., polynucleotides comprising an DnaJ homolog subfamily B member 5 (DNAJB5) gene or characteristic portion thereof, as well as compositions including such polynucleotides and methods utilizing such polynucleotides and/or compositions.
• a polynucleotide comprising an DNAIB5 gene or characteristic portion thereof can be DNA or RNA.
• DNA can be genomic DNA or cDNA.
• RNA can be an mRNA.
• a polynucleotide comprises exons and/or introns of an DNAJB5 gene.
• a gene product is expressed from a polynucleotide comprising an DNAJB5 gene or characteristic portion thereof.
• expression of such a polynucleotide can utilize one or more control elements (e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.).
• a polynucleotide provided herein can include one or more control elements.
• an DNAJB5 gene is a mammalian DNAJB5 gene.
• an DNAJB5 gene is a murine DNAJB5 gene.
• an DNAJB5 gene is a primate DNAJB5 gene.
• a DNAJB5 gene is a human DNAJB5 gene.
• An exemplary human DNAJB5 cDNA sequence is or includes the sequence of SEQ ID NO: 165 or 168 or 171.
• An exemplary human DNAJB5 genomic DNA sequence can be found in SEQ ID NO: 162.
• An exemplary human DNAIB5 cDNA sequence including untranslated regions is or includes the sequence of SEQ ID NO: 163, 164, 166, 167, 169, or 170.
• Exemplary Human DNAJB5 cDNA including untranslated regions Variant 4 (SEQ ID NO: 164)
• Exemplary Human DNAJB5 cDNA including untranslated regions Variant 2 (SEQ ID NO: 166)
• Exemplary Human DNAJB5 cDNA including untranslated regions Variant 6 (SEQ ID NO: 167)
• polynucleotide constructs include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno- associated viral constructs) that incorporate a polynucleotide comprising an NDP gene or characteristic portion thereof.
• cosmids e.g., naked or contained in liposomes
• viral constructs e.g., lentiviral, retroviral, adenoviral, and adeno- associated viral constructs
• polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno- associated viral constructs) that incorporate a polynucleotide comprising an HSPA1 A gene or characteristic portion thereof.
• cosmids e.g., naked or contained in liposomes
• viral constructs e.g., lentiviral, retroviral, adenoviral, and adeno- associated viral constructs
• polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno- associated viral constructs) that incorporate a polynucleotide comprising a gene encoding a secreted target protein (e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein), a HSPA1 A gene (e.g., any of the exemplary HSPA1 A genes described herein)) gene or characteristic portion thereof.
• a secreted target protein e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein)
• HSPA1 A gene e.g., any of the exemplary HSPA1 A genes described herein
• a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell).
• a construct can be a cosmid (e.g., pWE or sCos series).
• a construct is a viral construct.
• a viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct.
• a construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et ah, Mol. Ther. 20: 699-7080, 2012, which is incorporated in its entirety herein by reference).
• AAV adeno-associated virus
• a viral construct is an adenovirus construct.
• a viral construct may also be based on or derived from an alphavirus.
• Alphaviruses include Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O’nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus.
• viruses encode nonstmctural (e.g., replicon) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell.
• Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery.
• Pseudotyped viruses may be formed by combining alphaviral envelope glycoproteins and retroviral capsids. Examples of alphaviral constructs can be found in U S. Publication Nos. 20150050243, 20090305344, and 20060177819; constructs and methods of their making are incorporated herein by reference to each of the publications in its entirety.
• a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb.
• a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.
• a construct is a viral construct and can have a total number of nucleotides of up to 10 kb.
• a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 1 kb to about 10 kb,
• a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb.
• a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 2 kb to about 6
• a construct is an adenovirus construct and can have a total number of nucleotides of up to 8 kb.
• an adenovirus construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb
• any of the constructs described herein can further include a control sequence, e g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or additional untranslated regions which may house pre- or post-transcriptional regulatory and/or control elements.
• a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
• control sequences are described herein.
• the present disclosure provides AAV particles that comprise a construct encoding an NDP gene or characteristic portion thereof described herein, and a capsid described herein.
• the present disclosure provides AAV particles that comprise a construct encoding an HSPA1A gene or characteristic portion thereof described herein, and a capsid described herein.
• AAV particles that comprise a construct encoding a gene encoding a secreted target protein (e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein), a HSPA1 A gene (e.g., any of the exemplary HSPA1 A genes described herein)) gene or characteristic portion thereof described herein, and a capsid described herein.
• a secreted target protein e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein)
• HSPA1 A gene e.g., any of the exemplary HSPA1 A genes described herein
• AAV particles can be described as having a serotype, which is a description of the construct strain and the capsid strain.
• an AAV particle may be described as AAV2, wherein the particle has an AAV2 capsid and a construct that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs).
• ITRs characteristic AAV2 Inverted Terminal Repeats
• an AAV particle may be described as AAV1
• an AAV particle may be described as AAV2
• an AAV particle may be described as AAV3.
• an AAV particle may be described as AAV4.
• an AAV particle may be described as AAV5.
• an AAV particle may be described as AAV6.
• an AAV particle may be described as AAV7. In some embodiments, an AAV particle may be described as AAV8. In some embodiments, an AAV particle may be described as AAV9. In some embodiments, an AAV particle may be AAV-rh8. In some embodiments, an AAV particle may comprise an AAV-rhlO capsid. In some embodiments, an AAV particle may comprise an AAV-rh39 capsid. In some embodiments, an AAV particle may comprise an AAV-rh43 capsid. In some embodiments, an AAV particle may comprise an AAV Anc80 capsid.
• an AAV particle may be described as a pseudotype, wherein the capsid and construct are derived from different AAV strains as described herein, for example, AAV2/9 would refer to an AAV particle that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid.
• an AAV particle may be described as other AAV variants and serotypes as described herein.
• the present disclosure provides polynucleotide constructs that comprise a gene encoding a secreted target protein (e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein), a HSPA1 A gene (e.g., any of the exemplary HSPA1A genes described herein)) gene or characteristic portion thereof.
• a secreted target protein e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein)
• a HSPA1 A gene e.g., any of the exemplary HSPA1A genes described herein
• the present disclosure provides polynucleotide constructs that comprise an NDP gene or characteristic portion thereof.
• a polynucleotide comprising an NDP gene or characteristic portion thereof can be included in an AAV particle.
• the present disclosure also provides polynucleotide constructs that comprise an HSPA1A gene or characteristic portion thereof.
• a polynucleotide constmct comprises one or more components derived from or modified from a naturally occurring AAV genomic construct.
• a sequence derived from an AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV9 construct, an AAV2.7m8 construct, an
• AAV8BP2 construct an AAV293 construct, or AAV Anc80 construct.
• Additional exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8: 184-197, 2017; Li et al., Mol. Ther. 16(7): 1252-1260, 2008; Adachi et al., Nat. Commun. 5: 3075, 2014; Isgrig et al., Nat. Commun. 10(1): 427, 2019; and Gao et al., J. Virol. 78(12): 6381-6388, 2004; each of which is incorporated in its entirety herein by reference.
• provided constructs comprise coding sequence, e.g., an gene encoding a secreted target protein (e.g., an NDP gene, e.g., an HSPA1A gene) or a characteristic portion thereof, one or more regulatory and/or control sequences, and optionally 5’ and 3’ AAV derived inverted terminal repeats (ITRs).
• a secreted target protein e.g., an NDP gene, e.g., an HSPA1A gene
• ITRs optionally 5’ and 3’ AAV derived inverted terminal repeats
• the polynucleotide construct may be referred to as an AAV construct.
• provided AAV constructs are packaged into an AAV capsid to form an AAV particle.
• AAV derived sequences typically include the cis-acting 5’ and 3’ ITR sequences (see, e.g., B. I. Carter, in“Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp. 155 168, 1990, which is incorporated herein by reference in its entirety).
• Typical AAV2-derived ITR sequences are about 145 nucleotides in length.
• at least 80% of a typical ITR sequence e.g., at least 85%, at least 90%, or at least 95%) is incorporated into a construct provided herein.
• any of the coding sequences and/or constructs described herein are flanked by 5’ and 3’ AAV ITR sequences.
• the AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.
• polynucleotide constructs described in accordance with this disclosure and in a pattern known to the art are typically comprised of, a coding sequence or a portion thereof, at least one and/or control sequence, and optionally 5’ and 3’ AAV inverted terminal repeats (ITRs).
• ITRs AAV inverted terminal repeats
• provided constructs can be packaged into a capsid to create an AAV particle.
• An AAV particle may be delivered to a selected target cell.
• provided constructs comprise an additional optional coding sequence that is a nucleic acid sequence (e.g., inhibitory nucleic acid sequence), heterologous to the construct sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
• a nucleic acid coding sequence is operatively linked to and/or control components in a manner that permits coding sequence transcription, translation, and/or expression in a cell of a target tissue.
• an unmodified AAV endogenous genome includes two open reading frames,“cap” and“rep,” which are flanked by ITRs.
• exemplary AAV constructs similarly include ITRs flanking a coding region, e.g., a coding sequence (e.g., a secreted target protein gene, e.g., an NDP gene).
• exemplary AAV constructs similarly include ITRs flanking a coding region, e.g., a coding sequence (e.g., a secreted target protein gene, e.g., an HSPA1A gene).
• an AAV construct also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription, translation and/or expression in a cell transfected with the plasmid construct or infected with the virus produced by the disclosure.
• an AAV construct optionally comprises a promoter (shown in Figure 19B), an enhancer, an untranslated region (e.g., a 5’ UTR, 3’ UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g., an acceptor site, a donor site), a polyadenylation site (shown Figure 20B), or any combination thereof.
• a promoter shown in Figure 19B
• an enhancer an untranslated region
• an internal ribosomal entry site IVS
• splicing sites e.g., an acceptor site, a donor site
• polyadenylation site shown Figure 20B
• an AAV construct is a recombinant AAV construct or
• an AAV construct can include at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb.
• an AAV construct can include at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb.
• an AAV construct can include about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
• AAV constructs are typically composed of, at a minimum, a transgene or a portion thereof and a regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). Such an AAV construct is packaged into a capsid and delivered to a selected target cell (e.g., a cochlear hair cell).
• any of the constructs described herein can further include regulatory and/or control sequences, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or any combination thereof.
• a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
• control sequences are described herein.
• the AAV sequences of the vector typically comprise the cis-acting 5' and 3' ITR sequences (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168, 1990, the contents of which is hereby incorporated by reference herein in its entirety).
• Typical AAV ITR sequences are about 145 nucleotides in length.
• at least 75% of a typical ITR sequence e.g., at least 80%, at least 85%, at least 90%, or at least 95%) is incorporated into the AAV vector. The ability to modify these ITR sequences is within the skill of the art.
• any of the coding sequences described herein is flanked by 5' and 3' AAV ITR sequences in the AAV vectors.
• the AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.
• AAV vectors as described herein may include any of the regulatory elements described herein (e.g., one or more of a promoter, a polyadenylation (poly(A)) signal sequence, and an IRES).
• a promoter e.g., one or more of a promoter, a polyadenylation (poly(A)) signal sequence, and an IRES.
• poly(A) polyadenylation
• IRES an IRES
• the AAV vector is selected from the group consisting of: an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV2.7m8 vector, an AAV8BP2 vector, and an AAV293 vector.
• Additional exemplary AAV vectors that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8: 184-197, 2017; Li et al., Mo/. Ther. 16(7): 1252-1260; Adachi et al., Nat. Commun.
• an AAV vector provided herein includes or consists of a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 9, 10, 11, 12, or 13.
• the vector(s) is an adenovirus vector (see, e.g., Dmitriev et al. (1998) J Virol. 72: 9706-9713; and Poulin et al., J Virol 8: 10074-10086, 2010, each of which is incorporated in its entirety herein by reference).
• the vector(s) is a retrovirus (see, e.g., Maier et al. (2010 ) Future Microbiol 5: 1507-23, the contents of which is incorporated in its entirety herein).
• the vectors provided herein can be of different sizes.
• the choice of vector that is used in any of the compositions, kits, and methods described herein may depend on the size of the vector.
• the vector(s) can have a total number of nucleotides of up to 10 kb.
• the viral vector(s) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 2
• the vector(s) is a lentivirus and can have a total number of nucleotides of up to 8 kb.
• the lentivirus(es) can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb
• the vector(s) is an adenovirus and can have a total number of nucleotides of up to 8 kb.
• the adenovirus(es) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about
• the vector(s) is an adeno-associated virus (AAV vector) and can include a total number of nucleotides of up to 5 kb.
• AAV vector(s) can include a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
• a variety of different methods known in the art can be used to introduce any of vectors disclosed herein into a mammalian cell (e.g., an inner ear cell, a cochlear inner hair cell).
• methods for introducing nucleic acid into a mammalian cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.
• transfection e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrim
• Any of the vectors described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly A) signal, and a Kozak consensus sequence.
• a control sequence e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly A) signal, and a Kozak consensus sequence.
• a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
• AAV derived sequences of a construct typically comprises the cis-acting 5’ and 3’
• ITRs see, e g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated in its entirety herein by reference).
• ITRs are able to form a hairpin.
• the ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase-independent synthesis of a second DNA strand.
• ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.
• An rAAV particle (e.g., an AAV2/Anc80 particle) of the present disclosure can comprise a rAAV construct comprising a coding sequence (e.g., a gene encoding a secreted target protein or any characteristic portion thereof, e.g., an NDP gene or any characteristic portion thereof, e.g., an HSPA1A gene or any characteristic portion thereof) and associated elements flanked by a 5’ and a 3’ AAV ITR sequences.
• a coding sequence e.g., a gene encoding a secreted target protein or any characteristic portion thereof, e.g., an NDP gene or any characteristic portion thereof, e.g., an HSPA1A gene or any characteristic portion thereof
• an ITR is or comprises about 145 nucleic acids.
• all or substantially all of a sequence encoding an ITR is used.
• An AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types.
• An example of a construct molecule employed in the present disclosure is a“cis- acting” construct containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5’ or“left” and 3’ or“right” AAV ITR sequences.
• 5’ and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
• a 5’ or left ITR is an ITR that is closest to a promoter (as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
• 3’ and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
• a 3’ or right ITR is an ITR that is closest to a polyadenylation sequence (as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
• ITRs as provided herein are depicted in 5’ to 3’ order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5’ or“left” orientation ITR can also be depicted as a 3’ or“right” ITR when converting from sense to antisense direction.
• a given sense ITR sequence e.g., a 5’/left AAV ITR
• an antisense sequence e.g., 3’/right ITR sequence.
• One of ordinary skill in the art would understand how to modify a given ITR sequence for use as either a 5’/left or 3’/right ITR, or an antisense version thereof.
• an ITR (e.g., a 5’ ITR) can have a sequence according to SEQ ID NO: 1
• an ITR (e.g., a 3’ ITR) can have a sequence according to SEQ ID NO: 61, 63, 127, 138, or 152.
• an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, as is known in the art.
• an ITR comprises fewer than 145 nucleotides, e.g., 127,
• an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 ,124, 125, 126, 127, 128, 129, 130,
• an ITR (e.g., a 5’ ITR) can have a sequence according to SEQ ID NO: 62.
• an ITR (e.g., a 3’ ITR) can have a sequence according to SEQ ID NO: 63.
• a non-limiting example of a 5’ AAV ITR sequence is SEQ ID NO: 60.
• a non limiting example of a 3’ AAV ITR sequence is SEQ ID NO: 61.
• rAAV constructs of the present disclosure comprise a 5’ AAV ITR and/or a 3’ AAV ITR.
• a 5’ AAV ITR sequence is SEQ ID NO: 62.
• a 3’ AAV ITR sequence is SEQ ID NO: 63.
• the 5’ and a 3’ AAV ITRs flank a portion of a coding sequence, e.g., all or a portion of an NDP gene or HSPA1A gene (e.g., SEQ ID NO: 57 or 86).
• a coding sequence e.g., all or a portion of an NDP gene or HSPA1A gene (e.g., SEQ ID NO: 57 or 86).
• the ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al.“Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996), each of which is incorporated in its entirety herein by reference).
• a 5’ ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 5’ ITR sequence represented by SEQ ID NO: 60 or 62.
• a 3’ ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 3’ ITR sequence represented by SEQ ID NO: 61 or 63.
• Exemplary 3’ AAV ITR (SEQ ID NO: 61)
• Exemplary 5’ AAV ITR (SEQ ID NO: 62)
• Exemplary 3’ AAV ITR (SEQ ID NO: 63)
• promoter means a DNA sequence recognized by enzymes/proteins in a mammalian cell required to initiate the transcription of a specific gene (e.g., a secreted target gene (e.g., a NDP gene (e.g., any of the exemplary NDP genes described herein), a HSPA1 A gene (e.g., any of the exemplary HSPA1A genes described herein)).
• a promoter typically refers to, e.g., a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and at which transcription is initiated. Non-limiting examples of promoters are described herein. Additional examples of promoters are known in the art.
• a vector encoding an N-terminal portion of secreted target protein e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein), a HSPA1A protein (e.g., any of the exemplary HSPA1A proteins described herein) can include a promoter and/or an enhancer.
• the vector encoding the N-terminal portion of the secreted target protein can include any of the promoters and/or enhancers described herein or known in the art.
• the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art.
• the promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter.
• the promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter.
• the promoter will generally be one that is able to promote transcription in an inner hair cell
• the promoter is a cochlea-specific promoter or a cochlea- oriented promoter.
• promoters are known in the art that can be used herein.
• Non-limiting examples of promoters that can be used herein include: human EFla, human cytomegalovirus (CMV) (US Patent No. 5, 168,062), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), b-globin, b-actin, a-fetoprotein, g-globin, b- interferon, g-glutamyl transferase, mouse mammary tumor virus (MMTY), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g.
• human T-cell leukemia virus HTLV human T-cell leukemia virus HTLV
• AAV ITR interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), a-2- macroglobulin, vimentin, MHC class I gene H-2K b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HLA DQa and DC , interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), tropon
• promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007.
• the promoter is the CMV immediate early promoter.
• the promoter is a CAG promoter or a CAG/CBA promoter.
• the promoter is a CBA promoter, e.g., a CBA promoter comprising or consisting of SEQ ID NO: 18.
• the term“constitutive” promoter refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., a secreted target protein (e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein), a HSPA1 A protein (e.g., any of the exemplary HSPA1 A proteins described herein)), causes RNA to be transcribed from the nucleic acid in a mammalian cell under most or all physiological conditions.
• a protein e.g., a secreted target protein (e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein), a HSPA1 A protein (e.g., any of the exemplary HSPA1 A proteins described herein)
• constitutive promoters include, without limitation, the retroviral
• Rous sarcoma virus (RSV) LTR promoter Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al, Cell 41 :521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 -alpha promoter (Invitrogen).
• RSV Rous sarcoma virus
• CMV cytomegalovirus
• SV40 promoter the dihydrofolate reductase promoter
• beta-actin promoter the beta-actin promoter
• PGK phosphoglycerol kinase
• EF1 -alpha promoter Invitrogen
• Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
• Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.
• inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)- inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. U.S.A. 93:3346- 3351, 1996), the tetracycline-repressible system (Gossen et al, Proc. Natl. Acad. Sci. U.S.A.
• tissue-specific promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory proteins that bind to the tissue-specific promoter).
• the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.
• tissue-specific promoters include but are not limited to the following: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an alpha- myosin heavy chain (a-MHC) promoter, and a cardiac Troponin T (cTnT) promoter.
• TSG liver-specific thyroxin binding globulin
• PY pancreatic polypeptide
• PPY pancreatic polypeptide
• Syn synapsin-1
• MCK creatine kinase
• DES mammalian desmin
• a-MHC alpha- myosin heavy chain
• cTnT cardiac Tropon
• Additional exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter (Sandig et al., Gene Ther. 3 : 1002-1009, 1996), alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther. 7: 1503-1514, 1996), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep. 24: 185-196, 1997); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res. 11 :654-664, 1996), CD2 promoter (Hansal et al., J. Immunol.
• the tissue-specific promoter is a cochlea-specific promoter.
• the tissue-specific promoter is a cochlear hair cell-specific promoter.
• cochlear hair cell-specific promoters include but are not limited to: a ATOH1 promoter, a POU4F3 promoter, a LHX3 promoter, a MY07A promoter, a MY06 promoter, a a9ACHR promoter, and a alOACHR promoter.
• the promoter is an cochlear hair cell-specific promoter such as a PRESTIN promoter or an ONCOMOD promoter. See, e.g., Zheng et al., Nature 405: 149-155, 2000; Tian et al. Dev. Dyn. 231 : 199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol . 66: 99-115, 2009, each of which is incorporated by reference in their entireties herein.
• tissue-specific promoter is an ear cell specific promoter.
• a tissue-specific promoter is an inner ear cell specific promoter.
• inner ear non-sensory cell-specific promoters include but are not limited to: GJB2, GJB6, SLC26A4, TECTA, DFNA5, COCH, NDP, SYN1, GFAP, PLP, TAK1, or SOX21.
• a cochlear non-sensory cell specific promoter may be an inner ear supporting cell specific promoter.
• Non-limiting examples of inner ear supporting cell specific promoters include but are not limited to: SOX2, FGFR3, PROX1, GLAST1, LGR5, HES1, HES5, NOTCH1, JAG1, CDKN1A, CDKN1B, SOX10, P75, CD44, HEY2, LFNG, or SlOOb.
• provided AAV constructs comprise a promoter sequence selected from a CAG, a CBA, a CMV, or a CB7 promoter.
• the first or sole AAV construct further includes at least one promoter sequence selected from Cochlea and/or inner ear specific promoters.
• a promoter is an endogenous human ATOH1 enhancer- promoter as set forth in SEQ ID NO: 68.
• an enhancer-promoter sequence is at least 85%, 90%, 95%, 98% or 99% identical to enhancer-promoter sequence represented by SEQ ID NO: 18.
• a promoter is an endogenous human SLC26A4 immediate promoter as set forth in SEQ ID NO: 103 or 93. In certain embodiments, a promoter is an endogenous human SLC26A4 enhancer-promoter as set forth in SEQ ID NO: 47, 48 or 50.
• an enhancer-promoter sequence is at least 85%, 90%, 95%, 98% or 99% identical to a promoter or enhancer-promoter sequence represented by SEQ ID NO: 103, 93, 47,
• a promoter is a human SLC26A4 endogenous enhancer- promoter sequence comprised within SEQ ID NO: 47, 48, or 50.
• a promoter is a human LGR5 enhancer-promoter as set forth in SEQ ID NO: 51.
• an enhancer-promoter sequence is at least 85%, 90%, 95%, 98% or 99% identical to enhancer-promoter sequence represented by SEQ ID NO: 51.
• a promoter is a human LGR5 endogenous enhancer-promoter sequence comprised within SEQ ID NO: 51.
• a promoter is a human SYN1 enhancer-promoter as set forth in SEQ ID NO: 52.
• an enhancer-promoter sequence is at least 85%,
• a promoter is a human SYN1 endogenous enhancer-promoter sequence comprised within SEQ ID NO: 52.
• a promoter is a human GFAP enhancer-promoter as set forth in SEQ ID NO: 53.
• an enhancer-promoter sequence is at least 85%, 90%, 95%, 98% or 99% identical to enhancer-promoter sequence represented by SEQ ID NO:
• a promoter is a human GFAP endogenous enhancer-promoter sequence comprised within SEQ ID NO: 53.
• a vector can include an enhancer sequence.
• Enhancer refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., a secreted target protein (e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein), a HSPA1A protein (e.g., any of the exemplary HSPA1A proteins described herein))).
• a protein of interest e.g., a secreted target protein (e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein), a HSPA1A protein (e.g., any of the exemplary HSPA1A proteins described herein))
• Enhancer sequences 50-1500 basepairs in length
• Enhancer sequences generally increase the level of transcription by providing additional binding sites for
• an enhancer sequence is found within an intronic sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter).
• enhancers include a RSV enhancer, a CMV enhancer, and a SV40 enhancer.
• the CMV enhancer sequence comprises or consists of SEQ ID NO: 17.
• a construct comprises a CMV enhancer exemplified by SEQ ID NO: 69.
• an enhancer sequence is at least 85%, 90%, 95%, 98% or 99% identical to the enhancer sequence represented by SEQ ID NO: 69.
• an SV-40 derived enhancer is the SV-40 T intron sequence, which is exemplified by SEQ ID NO: 70.
• a an enhancer sequence is at least 85%, 90%, 95%, 98% or 99% identical to the enhancer sequence represented by SEQ ID NO: 70.
• CMV enhancer SEQ ID NO: 69
• Exemplary SV-40 synthetic intron (SEQ ID NO: 70)
• any of the constructs provided herein can include splice donor and/or splice acceptor sequences, which are functional during RNA processing occurring during transcription. In some embodiments, splice sites are involved in trans-splicing.
• any of the vectors provided herein can include a polyadenylation (poly(A)) signal sequence.
• poly(A) polyadenylation
• the poly(A) tail confers mRNA stability and transferability (Molecular Biology of the Cell, Third Edition by B. Alberts et ah, Garland Publishing, 1994).
• the poly(A) signal sequence is positioned 3’ to the nucleic acid sequence encoding the C-terminus of the secreted target protein (e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein), a HSPA1 A protein (e.g., any of the exemplary HSPA1A proteins described herein)).
• a NDP protein e.g., any of the exemplary NDP proteins described herein
• HSPA1 A protein e.g., any of the exemplary HSPA1A proteins described herein
• “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end.
• the 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000 (SEQ ID NO: 100)) added to the pre-mRNA through the action of an enzyme,
• adenine nucleotides e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000 (SEQ ID NO: 100)
• polyadenylate polymerase In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation (or poly(A)) signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
• the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.
• a“poly(A) signal sequence” or“polyadenylation signal sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3’ end of the cleaved mRNA.
• poly(A) signal sequences that can be used, including those derived from bovine growth hormone (bgh) (Woychik et ah, Proc. Natl. Acad. Sci. U.S.A.
• mice-P-globin mouse-a-globin
• human collagen human collagen
• polyoma virus Bactet al ., Mol. Cell Biol. 15(9):4783-4790
• HSV TK Herpes simplex virus thymidine kinase gene
• IgG heavy-chain gene polyadenylation signal US
• hGH human growth hormone
• SV40 poly(A) site such as the SV40 late and early poly(A) site (Schek et al., Mol. Cell Biol. 12(12):5386-5393, 1992, which is incorporated herein by reference in its entirety).
• the poly(A) signal sequence can be AATAAA.
• the AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATT AAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACT AAA, A AT AT A, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e g., WO 06/12414, which is incorporated herein by reference in its entirety).
• the poly(A) signal sequence can be a synthetic
• the poly(A) signal sequence is the polyadenylation signal of bovine growth hormone (SEQ ID NO: 23).
• the poly(A) signal sequence is the polyadenylation signal of soluble neuropilin-1 (sNRP) (AAATAAAATACGAAATG (SEQ ID NO: 46)) (see, e.g., WO 05/073384, which is incorporated herein by reference in its entirety).
• a poly(A) signal sequence comprises or consists of the SV40 poly(A) site.
• a poly(A) signal comprises or consists of SEQ ID NO: 72.
• a poly(A) signal sequence comprises or consists of bGHpA. In some embodiments, a poly(A) signal comprises or consists of SEQ ID NO: 71. Additional examples of poly (A) signal sequences are known in the art. In some embodiments, a poly (A) sequence is at least 85%, 90%, 95%, 98% or 99% identical to the poly(A) sequence represented by SEQ ID NO: 71 or 72.
• Exemplary bGH poly(A) signal sequence (SEQ ID NO: 71)
• TGTCAT TCTAT TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT TGGGAAGACAATA
• Exemplary SV40 poly(A) signal sequence (SEQ ID NO: 72)
• a vector encoding the C-terminal portion of the secreted target protein can include a polynucleotide internal ribosome entry site (IRES).
• An IRES sequence is used to produce more than one polypeptide from a single gene transcript.
• An IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenb erg, Mol. Cell. Biol. 8(3): 1103-1112, 1988, which is incorporated herein in its entirety by reference).
• IRES sequences known to those in skilled in the art, including those from, e.g., foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV).
• FMDV foot and mouth disease virus
• EMCV encephalomyocarditis virus
• HRV human rhinovirus
• HAV human immunodeficiency virus
• HAV hepatitis A virus
• HCV hepatitis C virus
• PV poliovirus
• the IRES sequence that is incorporated into the vector that encodes the C-terminal portion of a secreted target protein is the foot and mouth disease virus (FMDV) 2A sequence.
• a secreted target protein e.g., a NDP protein (e.g., any of the exemplary NDP proteins described herein)
• a HSPA1 A protein e.g., any of the exemplary HSPA1 A proteins described herein
• FMDV foot and mouth disease virus
• the Foot and Mouth Disease Virus 2A sequence is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et ah, EMBO 4:928-933, 1994; Mattion et ah, J. Virology 70:8124-8127, 1996; Furler et ah, Gene Therapy 8:864-873, 2001; and Halpin et ah, Plant Journal 4:453-459, 1999 , each of which is incorporated in its entirety herein by reference).
• the cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan et al., EMBO 4:928-933, 1994; Mattion et al., J.
• An IRES can be utilized in an AAV construct.
• a construct encoding the C-terminal portion of the secreted target protein can include a polynucleotide internal ribosome entry site (IRES).
• IRES polynucleotide internal ribosome entry site
• an IRES can be part of a composition comprising more than one construct.
• an IRES is used to produce more than one polypeptide from a single gene transcript.
• constructs provided herein can optionally include a sequence encoding a reporter polypeptide and/or protein (“a reporter sequence”).
• reporter sequences include DNA sequences encoding: a beta-lactamase, a beta- galactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a green fluorescent protein (GFP), a red fluorescent protein, an mCherry fluorescent protein, a yellow fluorescent protein, a chloramphenicol acetyltransferase (CAT), and a luciferase. Additional examples of reporter sequences are known in the art.
• the reporter sequence When associated with regulatory' elements which drive their expression, the reporter sequence can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays; fluorescent activating cell sorting (FACS) assays; immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).
• FACS fluorescent activating cell sorting
• immunological assays e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry.
• the reporter sequence is tGFP (SEQ ID NO: 27). In some embodiments, the reporter sequence is eGFP (SEQ ID NO: 28 or SEQ ID NO: 29). In some embodiments, the reporter sequence is the LacZ gene, and the presence of a vector carrying the LacZ gene in a mammalian cell (e.g., a cochlear hair cell, an ocular cell, such as a retinal cell) is detected by assays for beta-galactosidase activity.
• a mammalian cell e.g., a cochlear hair cell, an ocular cell, such as a retinal cell
• the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase
• a vector carrying the fluorescent protein or luciferase in a mammalian cell e.g., a cochlear hair cell, an ocular cell, such as a retinal cell
• fluorescent techniques e.g., fluorescent microscopy or FACS
• light production in a luminometer e.g., a spectrophotometer or an IVIS imaging instrument.
• the reporter sequence can be used to verify the tissue-specific targeting capabilities and tissue-specific promoter regulatory activity of any of the vectors described herein.
• a reporter sequence is the LacZ gene, and the presence of a construct carrying the LacZ gene in a mammalian cell (e.g., a cochlear hair cell) is detected by assays for beta-galactosidase activity.
• the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase
• the presence of a construct carrying the fluorescent protein or luciferase in a mammalian cell e.g., a cochlear hair cell
• a reporter sequence can be used to verify the tissue-specific targeting capabilities and tissue-specific promoter regulatory and/or control activity of any of the constructs described herein.
• a reporter sequence is a FLAG tag (e.g., a 3xFLAG tag), and the presence of a construct carrying the FLAG tag in a mammalian cell (e.g., an inner ear cell, e.g., a cochlear hair or supporting cell) is detected by protein binding or detection assays (e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry).
• An exemplary 3xFLAG tag sequence is provided as SEQ ID NO: 105.
• any of the vectors described herein can include an untranslated region, such as a 5’UTR or a 3’ UTR.
• UTRs Untranslated regions
• UTR starts at the transcription start site and continues to the start codon but does not include the start codon.
• a 3’ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
• the regulatory and/or control features of a UTR can be incorporated into any of the vectors, constructs, compositions, kits, or methods as described herein to otherwise modulate the expression of a secreted target protein (e.g., a NDP protein, a HSPA1 A protein).
• Natural 5’ UTRs include a sequence that plays a role in translation initiation.
• Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another“G”.
• R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another“G”.
• the 5’UTRs have also been known to form secondary structures that are involved in elongation factor binding.
• a 5’UTR is included in any of the vectors described herein.
• Non-limiting examples of 5’UTRs including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as an mRNA.
• a 5’UTR from an mRNA that is transcribed by a cell in the cochlea or retina can be included in any of the vectors, compositions, kits, and methods described herein.
• a 5’ UTR is derived from the endogenous SLC26A4 gene loci and may include all or part of the endogenous sequence exemplified by SEQ ID NO:
• a 5’ UTR sequence is at least 85%, 90%, 95%, 98% or 99% identical to the 5’ UTR sequence represented by SEQ ID NO: 21.
• 3’ UTRs are found immediately 3’ to the stop codon of the gene of interest.
• a 3’ UTR from an mRNA that is transcribed by a cell in the cochlea can be included in any of the constructs, compositions, kits, and methods described herein.
• a 3’ UTR is derived from the endogenous SLC26A4 gene loci and may include all or part of the endogenous sequence exemplified by SEQ ID NO: 22.
• a 3’ UTR sequence is at least 85%, 90%, 95%, 98% or 99% identical to the 3’ UTR sequence represented by SEQ ID NO: 22.
• 3’UTRs are known to have stretches of adenosines and uridines (in the RNA form) or thymidines (in the DNA form) embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al , Mol. Cell. Biol. 15:2010-2018, 1995 , each of which is incorporated herein by reference in its entirety): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping
• UUAUUUA(U/A) (U/A) nonamers UUAUUUA(U/A) (U/A) nonamers.
• GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs.
• Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif.
• Two well-studied examples of this class are c-Jun and myogenin mRNAs.
• HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3’UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
• An exemplary human wildtype 5’UTR is or includes the sequence of SEQ ID NO:
• An exemplary human wildtype 3’UTR is or includes the sequence of SEQ ID NO: 15 or 22.
• a 5’ untranslated region (UTR), a 3’UTR, or both are included in a vector (e.g., any of the vectors described herein).
• a vector e.g., any of the vectors described herein.
• any of the 5’UTRs described herein can be operatively linked to the start codon in any of the coding sequences described herein.
• any of the 3’UTRs can be operably linked to the 3’-terminal codon (last codon) in any of the coding sequences described herein.
• the 5’UTR includes at least 10 contiguous (e.g., at least 15 contiguous, at least 20 contiguous, at least 25 contiguous, at least 30 contiguous, at least 35 contiguous, at least 40 contiguous, at least 45 contiguous, at least 50 contiguous, at least 55 contiguous, at least 60 contiguous, at least 65 contiguous, at least 70 contiguous, at least 75 contiguous, at least 80 contiguous, at least 85 contiguous, at least 90 contiguous, at least 95 contiguous, at least 100 contiguous, at least 105 contiguous, at least 110 contiguous, at least 115 contiguous, at least 120 contiguous, at least 125 contiguous, at least 130 contiguous, at least 135 contiguous, at least 140 contiguous, at least 145 contiguous, at least 150 contiguous, at least 155 contiguous, at least 160 contiguous, at least
• a 5’UTR can include or consist of one or more of: nucleotide positions 1 to 550, nucleotide positions 1 to 500, nucleotide positions 1 to 450, nucleotide positions 1 to 400, nucleotide positions 1 to 350, nucleotide positions 1 to 300, nucleotide positions 1 to 250, nucleotide positions 1 to 200, nucleotide positions 1 to 150, nucleotide positions 1 to 100, nucleotide positions 1 to 50, nucleotide positions 50 to 550, nucleotide positions 50 to 500, nucleotide positions 50 to 450, nucleotide positions 50 to 400, nucleotide positions 50 to 350, nucleotide positions 50 to 300, nucleotide positions 50 to 250, nucleotide positions 50 to 200, nucleotide positions 50 to 150, nucleotide positions 50 to 100, nucleotide positions 100 to 550, nucleotide positions 100
• the 5’UTR includes a sequence that is at least 70% (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 14 or SEQ ID NO: 31.
• the 3’UTR includes at least 10 contiguous (e.g., at least 15 contiguous, at least 20 contiguous, at least 25 contiguous, at least 30 contiguous, at least 35 contiguous, at least 40 contiguous, at least 45 contiguous, at least 50 contiguous, at least 55 contiguous, at least 60 contiguous, at least 65 contiguous, at least 70 contiguous, at least 75 contiguous, at least 80 contiguous, at least 85 contiguous, at least 90 contiguous, at least 95 contiguous, at least 100 contiguous, at least 105 contiguous, at least 110 contiguous, at least 115 contiguous, at least 120 contiguous, at least 125 contiguous, at least 130 contiguous, at least 135 contiguous, at least 140 contiguous, at least 145 contiguous, at least 150 contiguous, at least 155 contiguous, at least 160 contiguous, at least 10 contiguous (e.g., at least 15 contiguous, at
• a 3’UTR can include or consist of one or more of: nucleotide positions 1 to 1000, nucleotide positions 1 to 950, nucleotide positions 1 to 900, nucleotide positions 1 to 850, nucleotide positions 1 to 800, nucleotide positions 1 to 750, nucleotide positions 1 to 700, nucleotide positions 1 to 650, nucleotide positions 1 to 600, nucleotide positions 1 to 550, nucleotide positions 1 to 500, nucleotide positions 1 to 450, nucleotide positions 1 to 400, nucleotide positions 1 to 350, nucleotide positions 1 to 300, nucleotide positions 1 to 250, nucleotide positions 1 to 200, nucleotide positions 1 to 150, nucleotide positions 1 to 100, nucleotide positions 1 to 50, nucleotide positions 50 to 1000, nucleotide positions 50 to 950, nucleotide positions 1 to 950
• the 3’UTR includes a sequence that is at least 70% (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 15 or SEQ ID NO: 22.
• the introduction, removal, or modification of 3’UTR AREs can be used to modulate the stability of an mRNA encoding a secreted target protein (e.g., a NDP protein, a HSPA1 A protein).
• a secreted target protein e.g., a NDP protein, a HSPA1 A protein
• AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of a secreted target protein (e.g., a NDP protein, a HSPA1 A protein).
• non- ARE sequences may be incorporated into the 5’ or 3’
• introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the vectors, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
• the vector includes a chimeric intron sequence (SEQ ID NO: 19).
• constructs of the present disclosure may comprise a T2A element or sequence.
• constructs of the present disclosure may include one or more cloning sites.
• cloning sites may not be fully removed prior to manufacturing for administration to a subject.
• cloning sites may have functional roles including as linker sequences, or as portions of a Kozak site.
• linker sequences or as portions of a Kozak site.
• constructs may contain any combination of cloning sites, exemplary cloning sites are represented by SEQ ID NO:73-77, 106, 75 and 107, respectively, in order of appearance.
• Exemplary cloning site A (SEQ ID NO: 73)
• Exemplary cloning site B (SEQ ID NO: 74)
• Exemplary cloning site C (SEQ ID NO: 75) TAAGAGCTCGCTGATCAGCCTCGA
• Exemplary cloning site D (SEQ ID NO: 76)
• Exemplary cloning site E (SEQ ID NO: 77)
• Exemplary cloning site F (SEQ ID NO: 106)
• any of the constructs provided herein can optionally include a sequence encoding a destabilizing domain (“a destabilizing sequence”) for temporal control of protein expression.
• a destabilizing sequence include sequences encoding a FK506 sequence, a dihydrofolate reductase (DHFR) sequence, or other exemplary destabilizing sequences.
• protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays; fluorescent activating cell sorting (FACS) assays; immunological assays (e.g., enzyme linked
• destabilizing sequences are known in the art.
• the destabilizing sequence is a FK506- and rapamycin-binding protein (FKBP12) sequence
• the stabilizing ligand is Shield-1 (Shldl) (Banaszynski et al. (2012) Cell 126(5): 995-1004, which is incorporated in its entirety herein by reference).
• a destabilizing sequence is a DHFR sequence
• a stabilizing ligand is trimethoprim (TMP) (Iwamoto et al. (2010) Chem Biol 17:981-988, which is incorporated in its entirety herein by reference).
• a destabilizing sequence is a FKBP12 sequence, and a presence of an AAV construct carrying the FKBP12 gene in a subject cell (e.g., a supporting cochlear outer hair cell) is detected by western blotting.
• a destabilizing sequence can be used to verify the temporally-specific activity of any of the AAV constructs described herein.
• DHFR destabilizing amino acid sequence SEQ ID NO: 1028
• DHFR destabilizing nucleotide sequence SEQ ID NO: 109
• FKBP12 destabilizing peptide amino acid sequence SEQ ID NO: 111) MGVEKQVIRPGNGPKPAPGQTVTVHCTGFGKDGDLSQKFWSTKDEGQKPFSFQIGKGAVIKGWD
• an AAV capsid is from or derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rhlO, rh39, rh43 or Anc80 serotype, or one or more hybrids thereof.
• an AAV capsid is from an AAV ancestral serotype.
• an AAV capsid is an ancestral (Anc) AAV capsid.
• An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence.
• an AAV capsid/construct sequence is not known to have existed in nature.
• an AAV capsid is an Anc80 capsid (e.g., an Anc80L65 capsid).
• an AAV capsid is created using a template nucleotide coding sequence comprising SEQ ID NO: 58.
• the capsid comprises a polypeptide represented by SEQ ID NO: 59.
• the capsid comprises a polypeptide with at least 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide represented by SEQ ID NO: 59.
• any combination of AAV capsids and AAV constructs may be used in recombinant AAV (rAAV) particles of the present disclosure.
• rAAV recombinant AAV particles of the present disclosure.
• AAV2 ITRs and Anc80 capsid wild type or variant AAV2 ITRs and AAV6 capsid, etc.
• an AAV particle is wholly comprised of AAV2 components (e.g., capsid and ITRs are AAV2 serotype).
• an AAV particle is an AAV2/6, AAV2/8 or AAV2/9 particle (e.g., an AAV6, AAV8 or AAV9 capsid with an AAV construct having AAV2 ITRs).
• an AAV particle is an AAV2/Anc80 particle that comprises an Anc80 capsid (e.g., comprising a polypeptide of SEQ ID NO: 58) that encapsulates an AAV construct with AAV2 ITRs (e.g., SEQ ID NOs: 60 and 61) flanking a portion of a coding sequence, for example, a gene encoding a secreted target protein (e.g., an NDP gene, e.g., an HSPA1A gene) or characteristic portion thereof (e.g., SEQ ID NO: 1, 33, 3, 33, or 85).
• an Anc80 capsid e.g., comprising a polypeptide of SEQ ID NO: 58
• AAV2 ITRs e.g., S
• a capsid sequence is at least 85%, 90%, 95%, 98% or 99% identical to a capsid nucleotide or amino acid sequence represented by SEQ ID NO: 8 or 9, respectively.
• AAV Anc80 Capsid Amino Acid Sequence (SEQ ID NO: 59) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEP LGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPP AAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHL YKQISSQSGASTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQ VKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNG SQAVGRSSFYCLEYFP
• compositions comprising a construct as described herein.
• a composition comprises one or more constructs as described herein.
• a composition comprises a plurality of constructs as described herein. In some embodiments, when more than one construct is included in the composition, the constructs are each different.
• a composition comprises an AAV particle as described herein. In some embodiments, a composition comprises one or more AAV particles as described herein. In some embodiments, a composition comprises a plurality of AAV particles. In come embodiments, when more than one AAV particle is included in the composition, the AAV particles are each different.
• a composition comprises secreted target protein. In some embodiments, a composition comprises a cell.
• a composition is or comprises a pharmaceutical composition.
• compositions or systems comprising AAV particles comprised of a single construct.
• a single construct may deliver a polynucleotide that encodes a functional (e.g., wild type or otherwise functional, e.g., codon optimized) copy of a gene encoding a secreted target protein (e.g., an NDP gene, e.g., an HSPA1A) or characteristic portion thereof.
• a construct is or comprises an rAAV construct.
• a single rAAV construct is capable of expressing a full-length secreted target protein (e.g., NDP, e.g., HSPA1 A) messenger RNA or a characteristic protein thereof in a target cell (e.g., an inner ear cell).
• a single construct e.g., any of the constructs described herein
• a single construct (e.g., any of the constructs described herein) can include a sequence encoding a functional secreted target protein (e.g., any construct that generates functional secreted target protein) and optionally additional polypeptide sequences (e.g., regulatory sequences, and/or reporter sequences).
• a functional secreted target protein e.g., any construct that generates functional secreted target protein
• additional polypeptide sequences e.g., regulatory sequences, and/or reporter sequences.
• a single construct composition or system may comprise any or all of the exemplary construct components described herein.
• an exemplary single construct is represented by SEQ ID NO: 9.
• an exemplary single construct is represented by SEQ ID NO: 10.
• an exemplary single construct is represented by SEQ ID NO: 11.
• an exemplary single construct is represented by SEQ ID NO: 12.
• an exemplary single construct is represented by SEQ ID NO: 13.
• an exemplary single construct is represented by SEQ ID NO: 96.
• an exemplary single construct is represented by SEQ ID NO: 97.
• an exemplary single construct is represented by SEQ ID NO: 98.
• an exemplary single construct is at least 85%, 90%, 95%, 98% or 99% identical to the sequence represented by SEQ ID NO: 9, 10, 11, 13, 96, 97, or 98.
• constructs may undergo additional modifications including codon-optimization, introduction of novel but functionally equivalent (e.g., silent mutations), addition of reporter sequences, and/or other routine modification.
• an exemplary construct comprises: a 5’ ITR exemplified by SEQ ID NO: 129, optionally a cloning site exemplified by SEQ ID NO: 129, a CMV enhancer exemplified by SEQ ID NO: 130, a CBA promoter exemplified by SEQ D NO: 131, a chimeric intron exemplified by SEQ ID NO: 132, optionally a cloning site exemplified by SEQ ID NO: 133, an NDP coding region exemplified by SEQ ID NO: 1 or an HSPA1A coding region exemplified by SEQ ID NO: 134 (optionally, wherein a IL2ss coding region precedes the HSPA1 A codon region exemplified by SEQ ID NO: 122), optionally a cloning site exemplified by SEQ ID NO: 135, a poly(A) site exemplified
• an exemplary construct comprises: a 5’ ITR exemplified by SEQ ID NO: 139, optionally a cloning site exemplified by SEQ ID NO: 140, a CMV enhancer exemplified by SEQ ID NO: 141, a CBA promoter exemplified by SEQ ID NO: 142, a chimeric intron exemplified by SEQ ID NO: 143, optionally a cloning site exemplified by SEQ ID NO: 144, an NDP coding region exemplified by SEQ ID NO: 1 or an HSPA1 A coding region as exemplified by SEQ ID NO: 145, optionally a reporter sequence exemplified by SEQ ID NO: 148, optionally a cloning site exemplified by SEQ ID NO: 149, a poly(A) site exemplified by SEQ ID NO: 150, optionally a cloning site
• compositions or systems comprising at least two different constructs (e.g., two, three, four, five, or six).
• each of the at least two different constructs includes a coding sequence that encodes a different portion of a coding region (e.g., encoding a target protein, e.g., an inner ear target protein, e.g., a secreted target protein), each of the encoded portions being at least 10 amino acids (e.g., at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids, at least about 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, at least about 180 amino acids, at least about 190 amino acids, at least about 200 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about
• one of the nucleic acid constructs can include a coding sequence that encodes a portion of a target protein (e.g., an inner ear target protein, e.g., a secreted target protein), where the encoded portion is at most about 820 amino acids (e.g., at most about 10 amino acids, at most about 20 amino acids, at most about 30 amino acids, at most about 60 amino acids, at most about 70 amino acids, at most about 80 amino acids, at most about 90 amino acids, at most about 100 amino acids, at most about 110 amino acids, at most about 120 amino acids, at most about 130 amino acids, at most about 140 amino acids, at most about 150 amino acids, at most about 160 amino acids, at most about 170 amino acids, at most about 180 amino acids, at most about 190 amino acids, at most about 200 amino acids, at most about 210 amino acids, at most about 220 amino acids, at most about 230 amino acids, at most about 240 amino acids, at most about 250 amino acids, at most about 260 amino acids, at most
• At least one of the constructs includes a nucleotide sequence spanning two neighboring exons of target genomic DNA (e.g., an inner ear target genomic DNA, e.g., NDP genomic DNA, e.g., HSPA1 A genomic DNA), and lacks the intronic sequence that naturally occurs between the two neighboring exons.
• target genomic DNA e.g., an inner ear target genomic DNA, e.g., NDP genomic DNA, e.g., HSPA1 A genomic DNA
• an amino acid sequence of an encoded portion of each of the constructs does not overlap, even in part, with an amino acid sequence of a different one of the encoded portions. In some embodiments, an amino acid sequence of an encoded portion of a construct partially overlaps with an amino acid sequence of an encoded portion of a different construct. In some embodiments, an amino acid sequence of an encoded portion of each construct partially overlaps with an amino acid sequence of an encoded portion of at least one different construct.
• an overlapping amino acid sequence is between about 10 amino acid residues to about 820 amino acids, or any of the subranges of this range (e.g., about 10 amino acids, about 20 amino acids, about 30 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 110 amino acids, about 120 amino acids, about 130 amino acids, about 140 amino acids, about 150 amino acids, about 160 amino acids, about 170 amino acids, about 180 amino acids, about 190 amino acids, about 200 amino acids, about 210 amino acids, about 220 amino acids, about 230 amino acids, about 240 amino acids, about 250 amino acids, about 260 amino acids, about 270 amino acids, about 280 amino acids, about 290 amino acids, about 300 amino acids, about 310 amino acids, about 320 amino acids, about 330 amino acids, about 340 amino acids, about 350 amino acids, about 360 amino acids, about 370 amino acids, about 380 amino acids, about 390 amino acids, about 400 amino acids, about 410 amino acids,
• a desired gene product (e.g., a therapeutic gene product) is encoded by at least two different constructs.
• each of at least two different constructs includes a different segment of an intron, where the intron includes a nucleotide sequence of an intron that is present in a target genomic DNA (e.g., an inner ear cell target genomic DNA (e.g., NDP genomic DNA, e.g., HSPA1 A genomic DNA) (e.g., any of the exemplary introns in SEQ ID NO: 3 described herein).
• a target genomic DNA e.g., an inner ear cell target genomic DNA (e.g., NDP genomic DNA, e.g., HSPA1 A genomic DNA)
• a target genomic DNA e.g., an inner ear cell target genomic DNA (e.g., NDP genomic DNA, e.g., HSPA1 A genomic DNA)
• a target genomic DNA e.g., an inner ear cell target genomic DNA (e.g.,
• different intron segments overlap in sequence by at most about 12,000 nucleotides (e.g., at most about 100 nucleotides, at most about 200 nucleotides, at most about 300 nucleotides, at most about 600 nucleotides, at most about 700 nucleotides, at most about 800 nucleotides, at most about 900 nucleotides, at most about 1,000 nucleotides, at most about 1,100 nucleotides, at most about 1,200 nucleotides, at most about 1,300 nucleotides, at most about 1,400 nucleotides, at most about 1,500 nucleotides, at most about 1,600 nucleotides, at most about 1,700 nucleotides, at most about 1,800 nucleotides, at most about 1,900 nucleotides, at most about 2,000 nucleotides, at most about 2,100 nucleotides, at most about 2,200 nucleotides, at most about 2,300 nucleotides, at most
• the overlapping nucleotide sequence in any two of the different constructs can include part or all of one or more exons of a target gene (e.g., an inner ear cell target gene (e.g., a secreted target protein (e.g., NDP, e.g., HSPA1A) gene) (e.g., any one or more of the exemplary exons in SEQ ID NO: 3 described herein).
• a target gene e.g., an inner ear cell target gene (e.g., a secreted target protein (e.g., NDP, e.g., HSPA1A) gene) (e.g., any one or more of the exemplary exons in SEQ ID NO: 3 described herein).
• a composition or system is or comprises two, three, four, or five different constructs.
• the first of the two different constructs can include a coding sequence that encodes an N-terminal portion of a protein (e.g., secreted target protein), which may be referred to as a lead portion, a first construct, or a 5’ portion (e.g., an N-terminal portion of an inner ear cell protein, e.g., an N-terminal portion of a secreted target protein).
• an N- terminal portion of the target gene is at least about 10 amino acids (e.g., at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids, at least about 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, at least about 180 amino acids, at least about 190 amino acids, at least about 200 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about 250 amino acids, at least about 260 amino acids, at least about 270 amino acids, at least about 280 amino acids, at least about 290 amino acids, at least about 300 amino acids, at least about 310 amino acids, at least about 320 amino acids, at least about 330 amino acids
• a first construct includes one or both of a promoter (e.g., any of the promoters described herein or known in the art) and a Kozak sequence (e.g., any of the exemplary Kozak sequences described herein or known in the art).
• a first construct includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
• a second of the two different constructs includes a coding sequence that encodes a C-terminal portion of the protein, which may be referred to as a terminal portion, a second construct, or a 3’ portion (e.g., a C-terminal portion of an inner ear cell target protein, e g., a C-terminal portion of a secreted target protein).
• a C-terminal portion of the target protein is at least about 10 amino acids (e.g., at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids, at least about 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, at least about 180 amino acids, at least about 190 amino acids, at least about 200 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about 250 amino acids, at least about 260 amino acids, at least about 270 amino acids, at least about 280 amino acids, at least about 290 amino acids, at least about 300 amino acids, at least about 310 amino acids, at least about 320 amino acids, at least about 330
• a second construct further includes a poly (A) sequence.
• an N-terminal portion encoded by one of the two constructs can include a portion including amino acid position 1 to about amino acid position 820, or any subrange of this range (e.g., amino acid 1 to at least about amino acid 10, amino acid 1 to at least about amino acid 20, amino acid 1 to at least about amino acid 30, amino acid 1 to at least about amino acid 60, amino acid 1 to at least about amino acid 70, amino acid 1 to at least about amino acid 80, amino acid 1 to at least about amino acid 90, amino acid 1 to at least about amino acid 100, amino acid 1 to at least about amino acid 110, amino acid 1 to at least about amino acid 120, amino acid 1 to at least about amino acid 130, amino acid 1 to at least about amino acid 140, amino acid 1 to at least about amino acid 150, amino acid 1 to at least about amino acid 160, amino acid 1 to at least about amino acid 170, amino acid 1 to at least about amino acid
• an N-terminal portion of the precursor inner ear cell target protein can include a portion including at most amino acid position 1 to amino acid position 820 or any subrange of this range (e g., amino acid 1 to at most about amino acid 10, amino acid 1 to at most about amino acid 20, amino acid 1 to at most about amino acid 30, amino acid 1 to at most about amino acid 60, amino acid 1 to at most about amino acid 70, amino acid 1 to at most about amino acid 80, amino acid 1 to at most about amino acid 90, amino acid 1 to at most about amino acid 100, amino acid 1 to at most about amino acid 110, amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 130 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 150 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 170 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 190 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 210 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 230 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 250 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 270 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 290 amino acid 1 to at most about amino acid 300
• amino acid 1 to at most about amino acid 310 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 330 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 350 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 370 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 390 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 410 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 430 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 450 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 470 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 490 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 510 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 530 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 550 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 570 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 590 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 610 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 630 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 650 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 670 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 690 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 710 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 730 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 750 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 770 amino acid 1 to at most about amino acid
• amino acid 1 to at most about amino acid 790 amino acid 1 to at most about amino acid
• an inner ear cell target protein e.g., SEQ ID NO: 6 or 7
• a C-terminal portion encoded by one of the two constructs can include a portion including the final amino acid (e.g., about amino acid position 820) to about amino acid position 1, or any subrange of this range (e g., amino acid 820 to at least about amino acid 10, amino acid 820 to at least about amino acid 20, amino acid 820 to at least about amino acid 30, amino acid 820 to at least about amino acid 60, amino acid 820 to at least about amino acid 70, amino acid 820 to at least about amino acid 80, amino acid 820 to at least about amino acid 90, amino acid 820 to at least about amino acid 100, amino acid 820 to at least about amino acid 110, amino acid 820 to at least about amino acid 120, amino acid 820 to at least about amino acid 130, amino acid 820 to at least about amino acid 140, amino acid 820 to at least about amino acid 150, amino acid 820 to at least about amino acid 160, amino acid 820 to at least about amino acid 170
• a C-terminal portion of the precursor inner ear cell target protein can include a portion including the final amino acid (e.g., about amino acid position 820) to at most about amino acid position 1, or any subrange of this range (e.g., amino acid 820 to at most about amino acid 10, amino acid 820 to at most about amino acid 20, amino acid 820 to at most about amino acid 30, amino acid 820 to at most about amino acid 60, amino acid 820 to at most about amino acid 70, amino acid 820 to at most about amino acid 80, amino acid 820 to at most about amino acid 90, amino acid 820 to at most about amino acid 100, amino acid 820 to at most about amino acid 110, amino acid 820 to at most about amino acid 120, amino acid 820 to at most about amino acid 130, amino acid 820 to at most about amino acid 140, amino acid 820 to at most about amino acid 150, amino acid 820 to at most about amino acid 160, amino acid 820 to at most about amino acid 170, amino
• splice sites are involved in trans-splicing.
• a splice donor site (Trapani et al. EMBO Mol. Med. 6(2): 194-211, 2014, which is incorporated in its entirety herein by reference) follows the coding sequence in the N-terminal construct.
• a splice acceptor site may be subcloned just before the coding sequence for NDP or HSPA1 A or other secreted target protein.
• a silent mutation can be introduced, generating an additional site for restriction digestion.
• any of the constructs provided herein can be included in a composition suitable for administration to an animal for the amelioration of symptoms associated with syndromic and/or non-syndromic hearing loss.
• the present disclosure provides pharmaceutical
• compositions are suitable for topical application.
• compositions provided herein are suitable for topical application.
• compositions of the present disclosure may comprise, e.g., a polynucleotide, e.g., one or more constructs, as described herein.
• a pharmaceutical composition may comprise one or more AAV particles, e.g., one or more rAAV construct encapsidated by one or more AAV serotype capsids, as described herein.
• a pharmaceutical composition comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into any of the compositions described herein.
• compositions may include one or more buffers, such as neutral-buffered saline, phosphate-buffered saline, and the like; one or more carbohydrates, such as glucose, mannose, sucrose, and dextran; mannitol; one or more proteins, polypeptides, or amino acids, such as glycine; one or more antioxidants; one or more chelating agents, such as EDTA or glutathione; and/or one or more preservatives.
• formulations are in a dosage forms, such as injectable solutions, injectable gels, drug-release capsules, and the like.
• compositions of the present disclosure are formulated for intravenous administration. In some embodiments compositions of the present disclosure are formulated for intra-cochlear administration. In some embodiments, a therapeutic composition is formulated to comprise a lipid nanoparticle, a polymeric nanoparticle, a mini -circle DNA and/or a CELiD DNA.
• a therapeutic composition is formulated to comprise a synthetic perilymph solution.
• a synthetic perilymph solution includes 20-200mM NaCl; 1-5 mM KC1; O.l-lOmM CaCl2; 1-lOmM glucose; and 2-50 mM HEPES, with a pH between about 6 and about 9.
• a therapeutic composition is formulated to comprise a physiologically suitable solution.
• a physiologically suitable solution comprises commercially available lxPBS with pluronic acid F68, prepared to a final concentration of: 8.
• any of the pharmaceutical compositions described herein may further comprise one or more agents that promote the entry of a nucleic acid or any of the constructs described herein into a mammalian cell (e.g., a liposome or cationic lipidj.
• any of the constructs described herein can be formulated using natural and/or synthetic polymers.
• Non-limiting examples of polymers that may be included in any of the compositions described herein can include, but are not limited to, DYNAMIC
• POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.), formulations from Mirus Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PhaseRX polymer formulations such as, without limitation, SMARTT POL YMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly (lactic-co- glycolic acid) (PLGA) polymers, RONDELTM (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, Calif.), and pH responsive co-block polymers, such as, but not limited to, those produced by PhaseRX (Seattle, Wash.).

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• 2020
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